1 WEIGHT OF EVIDENCE GUIDANCE: EVA LUATING 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 fo rth some of the general principles, criteria, and considerations EPA general ly 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 ac tivity under the Endocrine Disruptor Screening Program (EDSP). This paper was developed by EPA to provide guidance to EPA staff and managers who will be reviewin g data submitted in response Orders for Tier 1 screening that began October 29, 2009 under the Endocrine Disruptor Screening Program (EDSP). Additiona lly, 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 out side parties. As such, EPA may depart from the guidance where circumstances warrant and without prior notice.

A WoE evaluation is a proce ss where potentially relevant studies are judged in a professional manner for quality. Therea fter, a summary statement is developed indicating the potential effects of the co mpound, the mode of action (MOA), and other relevant information. It is not a process t hat simply involves tallying the number of positive and negative results withi n and among studies. Critic al assessment of an entire body of available data is taken into accoun t 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 indi vidual assays with multiple endpoints, as well as across the suite of a ssays in the Tier 1 scr eening battery. In addition, these principles would be generally rele vant to the review of other scientifically relevant information (OSRI) submitted in re sponse 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 discuss ed in this document are based on EPA’s experience in developing and applying risk assessment guidelines involving cancer, reproductive and development al toxicity, and ecological toxicity. Important considerations include the use of expert judgment formed through the scientific process, current understanding of endocrine mechan isms of toxicity, and knowledge of other fiel ds of toxicology ( e.g. , developmental, reproductive, neurological and immunological toxicology, and toxicoki netics). This document provides a transparent scientific approach for broadly evaluating Tier 1 screening data to determine if additional Tier 2 testing is necessary.2 This document also is expected to comp ly 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 u nder the EDSP and dire ction in the House Appropriations Committee for the Interior and Environment FY 2010 report (HR 2996, http://thomas.loc.gov/cgi-bin/c pquery/R?cp111:FLD010:@1(hr180)) that directed EPA

to:

“develop and publish criteria for evaluati ng the results of Tier 1 screening and determining whether a chemical should under go Tier 2 analysis wi thin one year of enactment. The process should allow for public input.”

2. HISTORICAL BACKGROUND

This section provides an abbreviated over view of EPA’s EDSP. A more detailed history of the program c an 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 t he Federal Food, Drug, and Cosmetic Act (FFDCA) to require EPA to:

“develop a screening program, using appr opriate validated test systems and other scientifically relevant information, to deter mine whether certain su bstances may have an
effect in humans that is similar to an effe ct produced by a naturally occurring estrogen,
or other such endocrine effect as the Admi nistrator may designate” [21 U.S.C. 346a(p)]. (http://www.epa.gov/pestici des/regulating/laws/fqpa/)

Fundamental to the EDSP is a two-tier ed 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 cons ists of a battery of complementary in vitro and

short term in vivo assays designed to maximize sensitiv ity 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-p ituitary-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 evaluati on 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-pit uitary-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 te sts are designed to quantify dos e-response relationships in a larger context of toxicity and potential adversity that may involve other biological3 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 screen ing 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 ev ents and processes, starting with interaction of an agent with a cell, proceeding through operational an d 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.4 Table 2. EDSP Ti er 2 tests.

Mammalian two-generation reproductive toxicity test

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

*Amphibian growth and development test - Lar val 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 vari ous stages of the validation process.

Most of the proposed Tier 1 screening assays complet ed the validation process in 2008 and another in 2009. Subsequent to review [Federal Regi ster Notice of January 24, 2008 (73 FR 4216)] by the Federal In secticide 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 (T able 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), summariz ed 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 fo r Series 890” at website). The endpoints associated with each assay were determi ned through the validation process to be sensitive and specific to detect interact ion 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 mechanisti c as well as some functional information on induction/inhibition of gene transcription or degree of ster oid hormone production, respectively.

In vivo assays integrate effects in a whole or ganism and provide apical as well as mechanistic information from one or multiple endpoints within an assa y. Agonistic or antagonistic E-, A-, or T- dependent changes can be detect ed in association with reproductive development ( e.g. , vaginal opening and preputial separation), organ weights ( e.g. , ovaries, uterus, testes, prosta te, and thyroid), and corresponding5 histology of target organs . Mechanistic information such as thyroid hormones measured in the pubertal and amphibian assa ys and vitellogenin in the fish assay can be correlated with apical in formation within the same assays. The use of gonadectomized rats in the ut erotrophic and Hershberger a ssays provides mechanistic information on specific estrogen- ( i.e. , uterus) or androgen- ( i.e. , prostate, seminal vesicles, levator ani-bulbocavernosus muscl e, 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, si nce more sites of action are involved along the HPG and HPT axes that target so me of the same E-, A-, or T-dependent endpoints.

3.2. Other Scientifically Re levant Information (OSRI)

EPA’s approach to the s ubmission and use of OSRI as part of the EDSP has previously been described by EPA and is av ailable in the Feder al Docket Management System (EPA-HQ-OPPT-2007-1080- 0032). In general, OSRI consists of data from assays that satisfy the same function as ED SP 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 m easured in standard toxicity test guidelines or other comparable toxicity studies. Typica lly, OSRI-types of studies are not designed to provide definitive information on the m odes or mechanisms of toxicity, but are generally focused on meas ured 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) repres enting permanent changes with or ganizational 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 typica lly be considered as part of EPA’s WoE approach:

• Do existing data provide adequate eviden ce to conclude whether there is a potential for the chemical to interfere wit h the normal function of the E, A, and/or T hormonal systems?

• If the data indicate a potential to intera ct with those specific endocrine systems, which hormone system is impacted (E, A, and/or T)?6 Determination of whether the evidence su ggests the substance is or is not a candidate for Tier 2 testing is based on evaluat ion 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, t he evaluation of individual studie s 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 t ransient changes; were the effects molecular/biochemical changes or adverse outcomes);

• specificity and sensitiv ity 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 di fferent effects seen within a study.

Both statistical and biological significance of the obs erved effects are relevant in evaluating study results. In general, the results of relev ant studies are assumed to be
indicative of interactions with the endocri ne 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 c onsequence of excessive toxicity to a nonendocrine system). To aid in determining the leve l of confidence in a study, the

s trengths 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 consi deration of the information as follows:

• Quality of data and the extent to wh ich effects can be replicated within a laboratory and across diffe rent 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 ev aluating the evidence as a whole and determining whether or not a chemical has the potential to interact with the endocrine7 system via E, A, and/or T horm onal pathways. In examining the balance of positive and negative results, the relative sensitivity and specific ity of the measur ed 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 physiologi cal conditions associated with whole tissues or organs and, therefor e, have nil or very limited ability to represent metabolic processes and pathways leadin g to endocrine disruption. The relationship between endpoints and their impact on normal endocri ne function would also typically be significant factors in this determination. Concordant effects found in multiple interrelated endpoints generally imply a compromi se in endocrine function, in contrast to

isolated or discordant effe cts. Totality of the evid ence is evaluated to determine whether such effects can potentially occur acro ss taxa. Generally, c onsistent positive or negative effects across studies and taxa incr ease confidence in the determination of

whether or not a chemical has the potential to interact with the endocrine system. Additionally, if marginal or weak relati onships 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 t hese 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 expe cted 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 charac terization 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

• i n consistent 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 ration ale for any conclusions. This may include, to the extent supported by the avail able data, conclusions regarding the species in which additional8 testing is warranted and the li kelihood that the effect may occur at a lower dose than effects seen in existing studies.9 REFERENCES EDSTAC (1998) Endocrine Disruptor Screeni ng 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 Envi ronmental 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/ma rch/minutes2008-03-25.pdf

.

USEPA (U.S. Environmental Protection Agen cy) (1991) Guidelines for developmental toxicity risk assessment. Federal Regi ster 56(234):63798-63826. Available from: http://cfpub.epa.gov/ncea/raf/r ecordisplay.cfm?deid=23162

.

USEPA (U.S. Environmental Protection Agen cy) (1996) Guidelines for reproductive toxicity risk assessment. Federal Regi ster 61(212):56274-56322. Available from: http://cfpub.epa.gov/ncea/raf/r ecordisplay.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/r ecordisplay.cfm?deid=12479

.

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

Reference Dose and Reference Concentrati on Processes. Risk Assessment Forum, Washington EPA/630/P-02/002F December 2002 Final Report http://www.epa.gov/raf/publicat ions/pdfs/rfd-final.pdf

USEPA (U.S. Environmental Protection Agency) (2005) Guidelines for carcinogen risk assessment Risk Assessment Forum, Wash ington, DC. EPA/630/P-03/001F. Available

from: http://www.epa.gov/raf/publications /pdfs/CANCER_GUI DELINES_FINAL_3-25- 05.PDFp://www.epa.gov/