EDSP: WEIGHT OF EVIDENCE ANALYSIS OF TIER 1 STUDIES

CASE STUDY: CHEMICAL X

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

OFFICE OF POLLUTION PREVENTION AND TOXICS

U.S ENVIRONMENTAL PROTECTION AGENCY
1. Case Study – Chemical X

A. Introduction

Chemical X is presented as a specific case study to illustrate the process of applying a Weight of 

Evidence (WoE) analysis to determine a chemical’s potential to interact with the endocrine 

system. Unlike the EDSP List 1 case studies, Chemical X was not subject to EDSP Tier 1 

screening and, therefore, only Other Scientifically Relevant Information (OSRI) is available for 

consideration in the WoE evaluation. Therefore this case study will focus on how OSRI may in 

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

Chemical X is a non-pesticide that comes from a family of closely related, commercially 

important alkylphenols used in the production of ethoxylates which have a wide variety of 

industrial applications and are produced in large volumes. The endocrine activity of Chemical X 

and its associated ethoxylates and carboxylates has been well studied in vitro and in vivo and, 

therefore, was chosen as a case study to illustrate the WoE process solely based on OSRI.

The OSRI chosen to represent the potential for Chemical X to interact with the endocrine system 

consists primarily of published, scientifically peer-reviewed studies from the public domain. 

When possible, the selection of studies was based on those that closely resembled in principle 

and methodology of the EDSP Tier 1 screening assays. It is emphasized that this case study, as 

with the EDSP List 1 case studies, is intended only to be illustrative of potentially challenging 

scenarios involved in the implementation of the WoE guidance. The agency is intentionally

keeping the identity of this chemical anonymous to avoid any chemical–specific 

recommendations and to ensure a broad interpretation of this case so that it may be generalized 

for other chemicals that might have a similar toxicity profile. The presentation of Chemical X to 

the SAP will not have any impact on the current regulatory standing of Chemical X. The OSRI 

selected and the WoE analyses presented for this case should not be interpreted as 

comprehensive, final or complete beyond this limited illustration. The agency will fully evaluate 

Chemical X, taking into account SAP comments on the WoE process, to determine whether or 

not Chemical X has the potential to interact with the endocrine system when the final SAP report 

is available. The SAP is asked to include Chemical X along with the other chemicals in their 

responses to respective charge questions.

B. Data Available for Chemical X

Chemical X is a phenolic compound with multiple isomers usually found as a pale yellow, 

highly viscous liquid, with an approximate molecular weight of 215.0 to 220.4 g/mole, specific 

gravity of 0.953 g/mL at 20°C (Budavari 1989), and a vapor pressure of 4.55 x 10-3(±3.54 x 10-

3

) Pa (Roy F. Weston Inc. 1990). It has a dissociation constant (pKa) of 10.7±1.0 and a log 

octanol/water partition coefficient (log Kow) of 3.80 to 4.77 (Roy F. Weston Inc. 1990). The 

water solubility of Chemical X is pH-dependent: 4,600 μg/L at pH 5.0; 6,237 μg/L at pH 7.0; 

11,897 μg/L at pH 9.0. The solubility of Chemical X in seawater is 3,630 μg/L and it is soluble 

in many organic solvents (Roy F. Weston Inc. 1990). Ahel and Giger (1993) measured the 

solubility of Chemical X at different temperatures in distilled water and demonstrated a nearly 

linear increase in solubility between 2°C (4,600 μg/L) and 25°C (6,350 μg/L). The chemical has 

and does not volatize appreciably and is characterized as being moderately persistent, as it is 
resistant (stable) to abiotic routes of degradation (i.e., hydrolysis and photolysis) and to 

anaerobic biotic metabolism. The relatively high log10 octanol-water partition coefficient and 

high organic carbon partition coefficient (Koc~60,000 mL/L), when combined with the 

compound’s low solubility, would limit the upper range of concentrations that could be tested. 

Although the compound is relatively lipophilic, it does not bioaccumulate appreciably in fish 

(BCF range 90 – 330x).

C. Other Scientifically Relevant Information (OSRI) for Chemical X

Information that is submitted voluntarily and is applicable to support or clarify an EPA action is 

generally referred to as “other” scientifically relevant information. Sources of relevant scientific 

and technical information may include toxicity study results from EPA or OECD equivalent test 

guidelines and information from published or publically available peer-reviewed studies. 

Regardless of the source, the information is evaluated for quality and relevance, in accord with 

EPA’s guidance on scientific integrity (EPA, 2012).

Chemical X has been widely investigated in the scientific community as having estrogenic 

activity; hence, the majority of published and publically available peer-reviewed studies

available as OSRI involve the estrogen hormonal pathway. OSRI that was selected to illustrate 

the WoE process applied to Chemical X consisted of in vitro and in vivo studies that are 

presented in a manner similar to the EDSP List 1 case studies for continuity. The results from 

OSRI for the various modes or pathways of action involving E, A and T for receptor binding 

(agonist and antagonist) and activation/transcription, steriodogenesis (competitive induction or 

inhibition), and the hypothalamic-pituitary-gonadal and -thyroidal axes are summarized in the 

following sections in context with respective EDSP Tier 1 screening assays.

i. ER Binding Assays

The results from several studies which are technically similar to the EDSP Tier 1 ER binding 

assay (OCSPP 890.1250) that have demonstrated the potential of Chemical X to bind with the 

estrogen receptor are presented. In a study by Danzo (1997), ER binding was observed using 

rabbit uterine cytosol incubated with 7 nM [

3H] 17β-estradiol alone overnight at 4oC or with

100-fold molar excess of radioinert 17β-estradiol to determine nonspecific binding, or 100 μM of 

Chemical X. Results indicated Chemical X (red box) inhibited (75% reduction) binding of 

tritiated (

3H)estradiol (E2) to the estrogen receptor as shown in Figure 1.
Figure 1. The mean ± standard error is shown. Percent inhibition of 3H estradiol binding to the 

estrogen receptor is determined for a variety of chemicals. On the far left, the specific binding of 

3H estradiol (*E) is shown (100%). Second from the left, unlabeled estradiol (E) results in 100% 

inhibition (i.e. 0% [

3H] 17β-estradiol bound to receptor). Chemical X (100 μM) is indicated by

the red box. The numbers in parentheses above the bars indicate the number of experiments in 

which samples were assayed in triplicate. Figure adapted from Danzo 1997.

Blair et al. (2000) investigated 188 different chemicals for their relative binding affinity (RBA) 

to 17β-estradiol. Chemical X obtained from five different sources competitively displaced 

radiolabled ligand from the ER with a mean IC50 of 2.4 to 4.7 x 10-6and a RBA of from 0.02 to

0.037 % as shown in Figure 2.
Figure 2. Demonstrated affinity of Chemical X (open triangles) for the ER appears to be 

parallel to the linear portion of the curve of natural ligand estradiol (closed circles). Figure 

adapted from Blair et al., 2000.

In an investigation by Satoh et al. (2001), human ERα binding was quantified using 

fluorescence-labeled 17β-estradiol, incubated with Chemical X for 2 hr at room temperature, 

Chemical X bound to ERα with an IC50 of 7.0 x 10-7M and an RBA of 2.22%.

Laws et al. (2000), in a large study including multiple in vivo experiments substantiated by in 

vitro ER binding and transcription activation, reported weak affinity of Chemical X for the rat 

uterine cytosolic ER as demonstrated by Ki of 0.67 μM compared to 0.4 nM for 17-β-estradiol.

Chemical X represents a variety of isomers with linear and branched side chains which affect the 

affinity of the chemical for the estrogen receptor. For this reason, comparisons across studies 

may be difficult. Despite these challenges, the results of estrogen receptor binding assays 

conducted in different cell backgrounds and following different methods indicate Chemical X

binds to the estrogen receptor under a variety of assays conditions.

Thus, based on the combined results, Chemical X is considered a positive for ER binding in

competitive receptor binding assays.
ii. ER Transcriptional Activation Assays

Balaguer et al (1999) utilized four reporter gene assays using different cell lines transfected with 

various ER constructs to investigate the ability of chemical X to initiate ER mediated 

transcriptional activation. For one assay, the authors used MCF-7 cells, with endogenous ERα

expression, transfected with an ER-responsive luciferase reporter. The authors also used a HeLa 

cell line containing endogenously expressed ER stably transfected with ERα, ERβ, or a chimeric 

ERα receptor along with a luciferase reporter gene. After 3 days pretreatment with steroid-free 

calf serum to reduce steroid background of the culture medium, cells were incubated for 16 hr 

with appropriate reagents and luciferase activity was measured. In all models, the Chemical X 

mixture tested was active.

Figure 3. Induction of luciferase activity in (A) a MCF-7 cell line (MELN) and (B) a HeLa cell 

line (ER α) based ER-mediated luciferase reporter gene assays. Results are expressed as a 

percentage of luciferase activity measured per well (mean ± SEM of triplicate wells). The 100% 

value represents the value obtained in presence of E2 10-8M (9500 arbitrary units/0.1 mg of 

protein). Estradiol (E2) is shown in open triangles and Chemical X is indicated by open circles. 

Adapted from Balaguer et al. 1999.

Bonefeld-Jorgensen et al. (2007) also demonstrated ER transcription activation in the MVLN 

cell line, which are MCF-7 cells stably transfected with a luciferase reporter. Chemical X was 

tested in three independent runs for agonist and antagonist activity at concentrations ranging 

from 10-8 to 10-4M. Chemical X produced a clear estrogenic response with an EC50 of 8.9 μM. 

No antagonist activity was observed when Chemical X was tested in the presence of 25 uM.
Figure 4. Dose–response ER transactivation of E2 and Chemical X. The graph on the left shows 

relative luminescence in MVLN cells exposed to E2 (0.05–500 pM, closed circles) and to 10-8 to 

10-4M Chemical X (open diamonds) for 24 hr. The graph on the right shows 

agonistic/antagonistic ER activity of Chemical X tested alone or in co-exposure with 25 pM E2, 

which was set to 1. Mean values are shown (n ≥ 3). *Significantly different from the respective 

solvent controls (cells + 0.1% DMSO; 25 pM E2 + 0.1% DMSO). Adapted from BonefeldJorgenssen et al. 2007.

Using an ER-mediated, chemical-activated luciferase reporter gene (ER-CALUX) expressed in 

human T47D breast cancer cells, Legler et al. (1999) determined that Chemical X was among the 

most potent chemicals tested with an EC50 of 260 nM.

White et al. (1994) tested the effect of 4-Chemical X in rainbow trout (Oncorhynchus mykiss)

hepatocytes and several human breast cancer cell lines. Chemical X increased vitellogenin gene 

expression in trout hepatocytes and transcriptional activity in human breast cancer cells 

cotransfected with mouse estrogen receptor and a reporter gene (EREBLCAT) at concentrations 

in the range of 10-6to 10-5 M.
Figure 5. ER-mediated gene induction in A) rainbow trout hepatocytes and B) MCF-7 human 

breast cancer cells. The graph on the left shows vitellogenin induction in rainbow trout 

hepatocytes and the graph on the right shows fold induction of a luciferase reporter gene linked 

to the ER stably transfected into MCF-7 cells in response to 10-5, 10-6, and 10-7 M concentrations 

of Chemical X. On both graphs, the response to 10-8E2 is indicated by the first bar on the graph. 

Adapted from White et al. 1994.

Though different reporter gene constructs and cell backgrounds may have different sensitivities 

to native ligand and Chemical X, a variety of ER transcription activation assays comparable to 

the Tier 1 ERTA or quantifying similar ER-mediated gene activation support the ability of 

Chemical X to bind the ER and induce gene expression.

Thus, based on the combined results, Chemical X is considered positive for estrogenic activity in 

ER transcriptional activation assays.

iii. AR Binding Assays

The potential of Chemical X to bind the androgen receptor was tested in a variety of assay

systems. Satoh et al. (2001) incubated human androgen receptor for 1 hr incubation at 4oC with 

unlabeled testosterone, followed by the addition of an anti-testosterone antibody and peroxidaselabeled steroid for an additional hour. After plates were washed, Chemical X and substrate were 

added and developed color was quantified. Chemical X did interact with the human AR used in 

the kits and the IC50 and relative binding affinity were determined to be 1.3 x 10-5 M and 

0.131%, respectively, though from the results, the authors could not determine if Chemical X 

acted as an AR agonist or antagonist.
Figure 6. Competition by

Chemical X for Testosterone 

Binding to Androgen Receptor. 

Ligand binding experiments 

were carried out using a Ligand 

Screening System-Androgen 

Receptor kit (Toyobo, Japan). 

The SD was less than 2.6% (n = 

5). Ordinates: % of inhibition = 

A – B/A (A and B were the 

fluorescence intensities in the 

absence and presence of 

competitor, respectively. Closed 

circles indicate the positive 

control, milbolerone. Closed triangles indicate Chemical X. Adapted from Satoh et al. 2001. 

Using yeast transformed with full length mammalian AR and a luciferase reporter plasmid 

containing two AREs and a prostate specific antigen promoter, Lee et al. (2003) reported 5 nM 

Chemical X treatment inhibited [3H]5-dihydrotestosterone (DHT) binding to the AR by 30% in 

HeLa cells transfected with a luciferase-linked mouse AR, though the response was not dosedependent. Further, in transient transfection assays, Chemical X inhibited AR transcriptional 

activity (IC50 = 2.6 μM) and was a more potent inhibitor of androgen-induced gene expression 

than recognized antagonist cyproterone acetate. The inhibitory effects of Chemical X on 

androgen receptor-mediated signaling is supported by the reported antiandrogenic effect of 

Chemical X in the range of 0.60–20 μM, on the induction of receptor activity by the androgen 

agonist R1881 with an IC50 of 14.1 μM, and a maximum inhibition (MI) of 56%. Though high 

concentrations of Chemical X can cause cytotoxicity, the authors report yeast cell growth 

(measured by absorbance at 600 nm) was not significantly reduced in the concentrations of 

Chemical X tested in this study. 

Chemical X has been shown to significantly reduce AR gene transcription in Chinese hamster 

(Cricetulus griseus) ovary cells transfected with human AR and a luciferase reporter (BonefeldJorgensen et al., 2007). The potential AR antagonism of several compounds was tested in cells 

treated with increasing concentrations of Chemical X (0.15 to 40 x 10-6M) in the presence of an 

AR agonist (0.1 nM R1881). AR binding was analyzed using a four parameter logistic curve fit 

and an IC50 was calculated. Chemical X elicited an antagonist response with an IC50 of 14.1 μM 

and a maximum inhibition of 56% at 10-6 M. Chemical X was determined to be cytotoxic to 

CHO cells at concentrations > 40 x 10-

6M. 

Figure 7. AR antagonism of Chemical X 

(open triangles) on R1881 action on CHO 

cells stably transfected with AR and a 

luciferase reporter. Adapted from 

Bonefeld-Jorgensen et al. 2007.
These studies demonstrate that Chemical X interacts with the androgen receptor in in vitro

assays, though it is important to note that steroidal estrogens and xenoestrogens will elicit a 

positive response in AR binding assays.

iv. Steroidogenesis Assays

Effects of Chemical X on steroidogenesis were examined in assay systems substantially different 

than the in vitro EDSP Tier 1 Steroidogenesis assay (OCSPP 890.1550). Leydig cells from adult 

male Sprague-Dawley rats were isolated and treated with a range of low concentrations (0.0011, 

0.0033, 0.0055, 0.011, 0.022 mg/L) and higher concentrations (0.11, 0.55, 1.1, 1.65, 2.2, 2.75, 

3.3, 5.5 mg/L) of Chemical X disolved in 0.1% dimethylsulfoxide (DMSO). Cell viability was 

tested by trypan blue exclusion. Testosterone production measured after 48 hr was significantly 

increased in the low concentrations and reduced at higher concentrations. Concentrations of 

Chemical X greater than 2.2 mg/L resulted in morphologic changes to Leydig cell cultures such 

as cell shrinkage and relaxation of cell junctions. The decrease in testosterone production may

be the result of an increased Leydig cell death at high concentrations.

Figure 8. Testosterone concentrations in media of Leydig cells treated with low concentrations 

(left) and high concentrations (right) of Chemical X. * P < 0.05, ** P < 0.01, compared with the 

control group (n = 6). Adapted from Gong and Ham 2005.

Kortnet et al. (2009) reported that 1, 10, and 50 μM concentrations of Chemical X produced 

time- and concentration-specific effects on the expression of steriodogenic enzyme mRNA 

quantified by real-time PCR in in vitro Atlantic salmon (Salmo salar) ovarian tissues exposed for 

3 and 7 days. Expression of steriodogenic acute regulatory protein (StAR), side-chain cleavage 

enzyme (P450scc), 3 β-hydroxysteroid dehydrogenase (3β-HSD), and 17α-hydroxylase/17, 20 

lyase (P450C17) decreased with exposure to Chemical X. Increases in aromatase (P450 CYP19) 

transcript following exposure to 10 μM concentrations of Chemical X paralleled elevated ovarian 

estradiol levels at the same exposure concentration. Tissue levels of ovarian testosterone and 11-

ketotestosterone were significantly elevated following 7 days of exposure to all test 

concentrations of Chemical X. 

Results suggest Chemical X may interfer with in vitro steroidogenesis.
v. Aromatase Assays 

Chemical X was tested in an EPA-funded study (RTI International, 2007) during the interlaboratory validation studies for the Tier 1 Aromatase assay guideline (OSCPP 890.1200). 

Briefly, Chemical X was incubated for 15 min with human recombinant aromatase and tritiated 

androstenedione (1-β [

3H(N)]-androst-4-ene-3,17-dione ([3H]ASDN)) dissolved in ethanol and 

aromatase activity was determined by measuring the amount of tritiated water produced. Four 

independent runs were conducted and each run included a full activity control, a background 

activity control, a positive control series (5, 15, and 25 μM) including a known inhibitor 

(aminoglutethimide), and a range of concentrations of the test chemical (5, 7.5, 10, 15, and 20 

μM) with 2 replicates per concentration. Chemical X inhibited aromatase activity with an IC50 

of 21 μM.

In a second study, aromatase activity was measured in a human choriocarcinoma cell line was 

measured by tritiated water production from 3H -androstenedione substrate. Chemical X was 

added to cells and incubated for 18 hr after which cells were washed and provided with 0.2 μCi 

[1β3H] androst-4-ene-3,17-dione and 10 nM unlabeled 4- 4-androstene-3,17-dione. After 2 hr 

of incubation, the assay was terminated and cell media was extracted and analyzed for tritiated 

water production as a measure of aromatase activity. All concentrations of Chemical X were 

tested in triplicate and run in three independent assays, each of which included a positive control, 

the known aromatase inhibitor, 4-androsten-4-ol-3,17-dione. Chemical X concentrations in the 

range of 10-9to 10-5M resulted in a maximum inhibition of 71% observed at 10-5M (BonefeldJorgensen et al. 2007).

Based on the results of these studies, Chemical X appears to be an in vitro inhibitor of aromatase. 

vi. Uterotrophic Assays

The results from several studies done in a uterotrophic assays similar in design and methodology

to the EDSP Tier 1 screening assay (OCSPP 890.1600) are presented with the interpretation that 

a statistically significant (P<0.05) increase in uterine weight relative to controls is indicative of a 

positive response. In a study by Laws et al. (2000), an experiment was designed to evaluate the 

effects of Chemical X on uterine weight in sexually immature females rats (prepubertal) at 6 and 

24 hours following the last of 3 daily doses (25, 50, 100, and 200 mg/kg) when administered by 

oral gavage for 3 days starting on PND 21-24. Positive controls were 17 β-estradiol (E2) and 

ethynyl estradiol (EE). Wet uterine weight (i.e., uterus and fluid in uterine lumen) expressed as 

percent of control weight was significantly increased in prepubertal animals at the 50 and 100 

mg/kg doses at both 6 and 24 hr after the final dose as shown in Figure 9.

In another similarly designed experiment from the same study (Laws et al., 2000), the effects of 

Chemical X on uterine weight when administered by subcutaneous injection or oral gavage were 

examined. Both dosing routes resulted in an increase in uterine weight, although results with 

oral gavage appeared to have a significant effect at a lower dose (> 50 mg/kg) than subcutaneous 

delivery (> 100 mg/kg) as shown in Figure 10.
Figure 9. Three-day uterotrophic assay in prepubertal rats: comparison of effects 6 and 24 h 

after the last dose. Wet uterine weight expressed as percent of control following exposure to 17-

-estradiol (E2) by sc injection, or to ethynyl estradiol (EE), or Chemical X (red box) by oral 

gavage. *Significant treatment effect by ANOVA or the Kruskal-Wallis Nonparametric Anova 

Test (p = 0.05) with comparison to the control by Dunnett’s Multiple Comparison Test, MannWhitney test, or Dunn’s Multiple Comparisons Test (p < 0.05). Adapted from Laws et al. 2000.

Figure 10. Three-day uterotrophic assay in prepubertal rats: comparison of effects sc injection 

or oral gavage dosing routes. Wet uterine weight expressed as percent of control following 

exposure to 17-b-estradiol (far left) by sc injection, or to ethynyl estradiol (EE; second data set 

from left), or chemical X (red box) by oral gavage. *Significant treatment effect by ANOVA or 

the Kruskal-Wallis Nonparametric Anova Test (p = 0.05) with comparison to the control by 
Dunnett’s Multiple Comparison Test, Mann-Whitney test, or Dunn’s Multiple Comparisons Test 

(p < 0.05). Adapted from Laws et al. 2000.

Again, in the same study by Laws et al. (2000), the effect of Chemical X on uterine weight in 

intact prepuberal and adult ovariectomized female rats was compared. Uterine weights in adult

ovariectomized females were significantly increased as shown in Figure 11, but at doses greater 

(100 mg/kg) than required to increase uterine weight in the prepubertal rats as shown in Figures

9 and 10.

Figure 11. Three-day uterotrophic assay in adult ovariectomized rats. Wet uterine weight 

expressed as percent of control following exposure to 17-b-estradiol (far left) by sc injection, or 

to ethynyl estradiol (EE; second data set from left), or chemical X (red box) by oral gavage. 

*Significant treatment effect by ANOVA or the Kruskal-Wallis Nonparametric Anova Test (p = 

0.05) with comparison to the control by Dunnett’s Multiple Comparison Test, Mann-Whitney

test, or Dunn’s Multiple Comparisons Test (p < 0.05). Adapted from Laws et al. 2000.

Chemical X was also used as a reference chemical in a multi-laboratory validation study during 

development of the EDSP Tier 1 Uterotrophic Assay. The results are summarized in an 

integrated summary report (OECD, 2003). Briefly, Chemical X was administered to adult 

ovariectomized rats at 15, 75, 125, 250 and 350 mg/kg/d via oral gavage for 3 consecutive days. 

A vehicle control group (corn oil) and positive controls (17α-Ethinyl Estradiol, EE) were 

included to evaluate estrogen agonist activity. All animals survived to the end of treatment in the 

15, 75 and 125 mg/kg dose-groups; however, extensive mortality in the 250 and 350 mg/kg dose 

groups (38 and 67%, respectively) indicated overt toxicity at these treatment levels. At lower 

doses, uterine weight was statistically increased in the 75 and 125 mg/kg/d treatment groups in 

all 4 participating laboratories.

Thus, based on the combined results of the Uterotrophic Assays similar in design and 

methodology to the EDSP Tier 1 screening assay, Chemical X is positive for estrogenic activity 

as indicated by a significant increase in uterine weight.
vii. Hershberger Assays

In a Hershberger assay that was designed similar to the EDSP Tier 1 screening assay (OCSPP 

890.1400), Freyberger et al. (2007) castrated male Wistar rats on PND 45 and allowed them to 

acclimate for 1 week prior to test chemical administration in two distinct experiments. A 160 

mg/kg dose of Chemical X was administered in a corn oil suspension orally and subcutaneously 

for 10 days. One day after the last treatment, animals were killed and ventral prostate (VP), 

seminal vesicles (SV), gland penis (GP), levator ani and bulbocavernosus muscles (LABC), and 

Cowper’s glands (COWS) were removed and weighed. The study design included a testosterone 

propionate treatment (TP, 0.4 mg/kg) as an androgenic stimulus for testing antiandrogenic 

potential and flutamide (FLU, 3 mg/kg) as an antiandrogenic positive control. In accord with the 

OSCPP 890.1400 test guideline, a statistical increase (P<0.05) in the weights of two or more 

target tissues relative to controls is indicative of a positive response. Chemical X exposure did 

not result in a significant androgenic or an antiandrogenic response in this study.

Chemical X was also used as a reference chemical in a multi-laboratory validation study during 

development of the EDSP Tier 1 Hershberger Assay. The results are summarized in an 

integrated summary report (OECD, 2007). Briefly, Chemical X was administered at 160 mg/kg/d 

via oral gavage using the castrated rat model. A vehicle control group as well as positive control 

groups to evaluate androgen agonist (TP) and antagonist (FLU) activities were included.

Pooled across multiple laboratories, there was a decrease (P<0.01) in mean body weight 

(approximately 4%) for Chemical X compared to the vehicle control, thus indicating that 160 

mg/kg/d corresponded to a maximally tolerable dose (MTD). For the androgenic response, there 

was no statistically significant (p<0.05) increase in any of the 5 targeted tissues for Chemical X 

compared to the control group among the 10 laboratories. For the anti-androgenic response, 5 of 

the laboratories reported no significant decrease in any of the target tissue weights compared to 

the control. For the other 5 laboratories, a statistically significant decrease was detected in one 

or more target tissues. Notably, for those laboratories in which only one target tissue was 

affected, the tissue was different in each of the 4 laboratories. In only 1 of the 5 laboratories was 

a significant decrease detected in two of the target tissues that would be indicative of a positive 

response.

Thus, based on the combined results of the Hershberger Assays similar in design and 

methodology to the EDSP Tier 1 screening assay, androgenic activity of Chemical X was 

negative. 

viii. Pubertal Female Assay

The results for the pubertal female assays are represented by some studies in which the design 

and methodology was comparable to the EDSP Tier 1 screening assay (OCSPP 890.1450) and 

for others the results are from the offspring of generational and multigenerational studies. In a 

study by Kim et al. (2002) prepubertal female Sprague-Dawley rats were dosed for 20 days 

beginning on PND 21 to 10, 50, and 100 mg/kg of Chemical X delivered by oral gavage. 

Estrogenic activity of Chemical X was indicated by a significant advance in age at vaginal 

opening (VO). Irregular estrous cyclicity was characterized by increases in time to diestrus
following exposure to Chemical X at 50 and 100 mg/kg/d treatment. Ovarian weight was 

significantly decreased among females exposed to the high dose (100 mg/kg/d). Thyroid effects 

were indicated by a dose-dependent decrease in serum T4.

In a study by Nagao et al. (2000), Sprague-Dawley rats were dosed from PND 1 to 5 with 500 

mg/kg/day of Chemical X delivered subcutaneously with animals examined through puberty. 

All treated females experienced irregular estrous cycles with shortened diestrus. Increased 

atretic follicles and decreased corpora lutea were observed in ovaries and increased incidence of 

uterine histopathology was observed among treated females. Copulation and fertilization rates 

among male:female pairs were significantly reduced when compared to controls, as were the 

number of live embryos born to dams treated with Chemical X in the early postnatal period.

In a study by Laws et al. (2000), female Long-Evans rats were dosed from PND 21 to 35 with 

25, 50, and 100 mg/kg of Chemical X delivered by oral gavage. Age at vaginal opening was 

significantly advanced (i.e., the age of animals at VO decreased) following treatments of 50 

mg/kg and greater. The number of estrous cycles was significantly decreased following 100 

mg/kg Chemical X dosing as shown in Table 1, although the same dose failed to alter vaginal 

cytology of ovariectomized females following 11 days of exposure and only affected estrus 

cyclicity of adult, intact females following longer exposures (25 days) of 100 mg/kg of Chemical 

X administrated orally.

Table 1. Adapted from Laws et al. 2000.
In a multi-generation study, Nagao et al. (2001) treated adult female Sprague-Dawley rats 

beginning at 13 weeks of age with 2, 10, or 50 mg/kg of Chemical X delivered by oral gavage. 

Females of the F0 (parent) generation were treated from two weeks prior to pairing until 

necropsy (21 days after delivery of the F1 generation). First generation (F1) females were dosed 

indirectly through lactation and directly from weaning (PND 21) until necropsy (21 days after 

delivery of the F2 generation) and the F2 generation was terminated at weaning (PND 21). 

Chemical X dosing was continuous from the initiation of F0 generation through necropsy of the 

F2 animals. Neonatal survival (i.e., number of live F1 and F2 pups per litter) was significantly

reduced in the high treatment group (50 mg/kg), although postnatal growth was not affected. 

Treatment did not significantly affect estrous cycles or result in histopathological lesions in any

reproductive tissues examined. Absolute and relative uterine weights were significantly

increased among females exposed to 2 mg/kg and significantly decreased in the 50 mg/kg group. 

Ovarian weight was significantly decreased and vaginal opening was significantly advanced in 

the high treatment group, although treatment did not have an effect on estrous cycles, mating, or 

fertility. On postnatal day 22, serum concentrations of LH and FSH were decreased and T3 

increased.

In a multi-generation study by Chapin et al. (1999), Sprague-Dawley rats were treated with 200, 

650, and 2000 ppm of Chemical X via the diet. Calculated dose rates for young rats were 9 to 35 

mg/kg/d, 30 to 100 mg/kg/d, and 100 to 350 mg/kg/d, for each of the treatment groups 

respectively. Adult and PND 21 animals were necropsied. Vaginal opening was advanced (i.e.,

age decreased) in all generations and uterine weights were increased in the mid and high dose 

groups for only the F1 generation.

Thus, based on the combined results in female pubertal and multi-generational studies, Chemical 

X is positive for estrogenic activity as indicated by significant changes in various estrogendependent endpoints. The results on thyroid hormones was inconsistent.

ix. Pubertal Male Assay

The results for the pubertal male assays are represented by some studies in which the design and 

methodology was comparable to the EDSP Tier 1 screening assay (OCSPP 890.1500) and for 

others the results are from the offspring of generational and multigenerational studies. 

In a study by Benjamin et al. (2003), juvenile male Sprague-Dawley rats were exposed from 

PND 23 through 52/53 to 100 mg/kg/d. Chemical X significantly delayed pubertal onset, 

increased incidence of histopathology observed in the testis, and affected spermatogenesis in 

treated males.

In a study by Nagao et al. (2000), Sprague-Dawley rats were treated from PND 1 to 5 with 500 

mg/kg/d of Chemical X subcutaneously and examined animals through puberty. No significant 

alterations to behavior or plasma testosterone were observed and the increased time to preputial 

separation was not significantly altered from control. Chemical X-treated rat body weight was 

significantly reduced in males (though reductions were < 10% terminal body weight), however, 

relative testis weight was also significantly reduced. Chemical X exposure may have altered 

male fertility as indicated by gonadal histopathological examination. There was a decrease in 

germ cells in the seminiferous tubules and increased degenerate germ cells in the epididymides.
In a study by Nagao et al. (2001), Sprague-Dawley male rats were treated beginning at 6 weeks 

of age with 2, 10, or 50 mg/kg of Chemical X delivered by oral gavage. Males were treated for 

12 weeks prior to pairing. No adverse effects of Chemical X were observed on reproduction or 

sperm characteristics, and no histopathological lesions were observed in reproductive tissues. 

No significant differences were observed in relative reproductive organ weight, although the 

absolute weight of the epididymides of the 2 mg/kg treatment group were significantly greater 

than controls. Relative thyroid and pituitary gland weights were elevated among the high 

treatment groups. Preputial separation and anogenital distance were not affected. No 

histopathological lesions were observed in reproductive tissues; however, there was a significant 

increase in testosterone. Histologically, the thyroid gland appeared enlarged in the 50 mg/kg 

treatment group, and there was a dose dependent increase in TSH and a significant decrease in T3

and T4 in the 2 mg/kg group.

In a study by Gong and Han (2006), adult male Sprague-Dawley rats were treated from PND 35 

to 37 days with 125 and 250 mg/kg/d of Chemical X administered by oral gavage for 50 days 

and measured serum testosterone and luteinizing hormone.. Terminal samples indicated 

testosterone levels had dramatically declined in the high dose group, while LH increased in the 

125 and 250 mg/kg/d dose groups.

DeJager et al. (1999a) reported on the effects of Chemical X on male fertility after direct 

exposure to adult Sprague-Dawley rats in an OECD One-Generation Reproductive Toxicity

Study (OECD Test Guideline 415, 1983). Adult rats (20 animals/group) were treated daily via 

oral gavage with cotton seed oil (control) or Chemical X at 100, 250, and 400 mg/kg beginning 

at 12 weeks of age and ending at 22 weeks. The 10-week treatment period was expected to 

extend beyond the duration of normal spermatogenesis and epididymal transit time to cover at 

least one complete spermatogenic cycle (i.e., approximately 52 days). Endpoints included body

mass, testicular and epididymal mass, cauda epididymal sperm count, and testicular histology. A 

dose-dependent decrease in survival was observed; 0 deaths in the control, 3 deaths in the low-, 

15 deaths in the mid- and 18 deaths in the high-dose groups. There was no significant difference 

in body mass change over the duration of the study between males treated with Chemical X and 

controls as shown in Table 2. Testicular mass and testis mass:body mass ratio was significantly 

reduced (36 and 18%, respectively) in the high-dose group compared to the control group.

Epididymal mass and ratio was significantly reduced in the mid- (12 and 10%, respectively) and 

high-dose (56 and 44%) groups compared to controls. Total caudal epididymal sperm count was 

significantly reduced (79%) in the high-dose group compared to controls. Testicular histology

indicated normal spermatogenesis encompassing all 14 stages in all treatment groups; however, 

seminiferous tubule diameters were significantly reduced (10, 14, and 24%) in the low-, mid-, 

and high-dose groups, respectively, compared to the control group. Additionally, although not 

indicated in Table 2, seminiferous lumen diameter and epithelium thickness were significantly

reduced and thickened, respectively, in all treatment groups compared to the control group. The 

extreme loss of life in the mid- and high-dose groups is indicative of overt toxicity; therefore, the 

low-dose group was the only group to allow a reliable assessment of Chemical X on male 

fertility. Direct exposure of 100 mg/kg Chemical X to adult male rats resulted in a significant 

decreased in weight of the testis and epididymis and number of sperm.
Table 2. Adapted from DeJager et al., 1999a.

In another study, DeJager et al. (1999b) reported on the effects of Chemical X on male fertility

after gestational, lactational and direct exposure to neonatal and post-natal Sprague-Dawley rats 

in an OECD One-Generation Reproductive Toxicity Study (OECD Test Guideline 415, 1983). 

Dams (10 animals/group) were treated daily via oral gavage with cotton seed oil (control) or 

Chemical X at 100, 250, and 400 mg/kg beginning at Day 7 of gestation and ending at weaning 

(approximately 3 weeks after birth). After weaning, male pups (20 animals/group) were directly

exposed to Chemical X by oral gavage for 10 weeks to cover at least one complete 

spermatogenic cycle (i.e., approximately 52 days). Endpoints included body mass, testicular and 

epididymal mass, cauda epididymal sperm count, and testicular histology.
Table 3. Adapted from DeJager et al., 1999b.

No offspring were born from dams in the high-dose group (400 mg/kg), though all other groups 

had 100% survival of the first generation of male pups at the end of the treatment period. Body 

mass, represented by the difference in body mass from weaning to end of experimental period, 

was significantly decreased relative to control males among the low- (approximately 10%) and 

mid- (approximately 20%) dose groups. Testicular mass was significantly reduced in the lowand mid-dose group compared to the control group. Epididymal mass was significantly reduced 

in the mid-dose groups compared to controls. Overall analysis of the epididymal ratio was not 

significantly different among groups. Total caudal epididymal sperm count was significantly

reduced in the mid-dose group compared to controls. Testicular histology indicated normal 

spermatogenesis encompassing all 14 stages in all treatment groups. Seminiferous tubule 

diameters were significantly reduced in the low- and mid-dose groups compared to the control 

group. Additionally, although not indicated in Table 3, seminiferous lumen diameter and 

epithelium thickness were significantly reduced and thickened, respectively, in the low- and middose groups compared to the control group. Indirect and direct exposure to Chemical X during 

gestational, lactational and pubertal/peripubertal development impaired general growth that may 

be considered overtly toxic at 20% in the mid-dose group. Nonetheless, the negative impact on 

testicular and epididymal endpoints male is indicate of the effect of Chemical X on male fertility

in rats.

Thus, Chemical X is considered positive either as estrogenic or anti-androgenic in the adult male 

rat. Though many endpoints altered by Chemical X in the studies described in this section are 

not specific to either the E or A pathway, results strongly suggest the potential of chemical X to 

interact in the male rat endocrine system.
x. Fish Endocrine Relevant Studies

Harries et al. (2000) performed a shortterm (6 week) reproductive 

performance test with 4-month-old 

fathead minnow (Pimephales promelas) 

breeding pairs exposed to nominal 

treatment concentrations of 1, 10 and 

100 μg/L of chemical X (4 breeding 

pairs in two tanks, with 2 breeding pairs 

per tank separated by a perforated 

stainless steel partition) or fresh water 

(3 breeding pairs) and solvent (1 

breeding pair exposed to 0.2 mL/L

methanol) controls. Replicate tanks 

were 30 L in volume with a flowthrough and input of 0.5 L/min. 

Reproductive performance of each 

breeding pair was measured for 3 weeks 

prior to exposure to the test chemical 

and for 3 weeks during exposure to the 

test chemical. Mean total number of 

eggs spawned per pair was significantly

reduced in the highest Chemical X 

concentration (100 μg/L) compared to 

controls (Figure 12A) for the exposure 

period, but there were no significant 

differences between breeding pairs in 

the pre-exposure period. 

The mean number of spawnings per 

breeding pair (frequency of spawning) 

also showed a significant reduction in 

response to 10 and 100 μg/L Chemical 

X as compared to the control (Figure 

12B). The highest dose of Chemical X 

also produced a significant reduction in 

mean egg batch size per breeding pair 

(Figure 12C). 

Figure 12. A) Number of eggs spawned; B) number of spawnings; and C) egg batch size by

pairs of fathead minnow (Pimephales promelas) exposed to Chemical X. Data are presented as 

the mean number of eggs spawned ± SEM for 3 week periods prior to (open bars) and during 

(solid bars) exposure. Significant differences between the two periods are shown (* P < 0.05). 

Dilution water controls (Control), n=3 breeding pairs; solvent control, n=1 breeding pair; 

Chemical X, n=4 breeding pairs for each treatment. Adapted from Harries et al. 2000. 
Figure 13. Gonadosomatic index and plasma 

vitellogenin concentrations in fathead 

minnow (Pimephales promelas) after 3 

weeks exposure to Chemical X. Data are 

presented as means ± SEM. Significant 

differences between treatments and dilution 

water (*) and solvent (Ø) controls are shown 

(* P < 0.05). Dilution water controls 

(Control), n=3 breeding pairs; solvent 

control, n=1 breeding pair; Chemical X, n=4 

breeding pairs for each treatment. Adapted 

from Harries et al. 2000. 

Male fathead minnows showed a significant dose response increase to Chemical X as compared 

to dilution control water (control) in VTG levels but there were no significant differences in 

gonadosomatic index (Figure 13). 

To summarize, Harries et al. (2000) observed a clear dose response effect of Chemical X 

resulting in a decreased number of spawnings and an increase in male fathead minnow VTG 

plasma levels compared to controls. Alterations in these two endpoints were not seen at 1 μg/L 

Chemical X, but were seen at 10 and 100 μg/L Chemical X. Also the highest dose (100 μg/L) 

showed a significant reduction in number of eggs spawned and mean egg batch size.

Yokota et al. (2001) performed a reproduction study over two generations (F0 and F1) using 

medaka (Oryzias latipes) with exposure to control, solvent control (100 μL/L ethanol), and 1.85, 

5.56, 16.7, 50, and 150 μg/L Chemical X treatments. Exposure to the parental generation (F0) 

was from 24 hours post fertilization (hpf) to 104 days post hatch (dph) with a flow through 

system providing a 14 volume/day renewal rate to 1.8 L replicate test chambers. There were 4 

replicate test chambers from initiation (24 hpf) until 70 dph and each replicate began with 15 

embryos. At 60 dph, 5 larvae per replicate were sampled for secondary sex and histological 

analyses. At 70 dph, 6 phenotypic male:female mating pairs of F0 fish were established and eggs 

from spawning pairs were collected daily from 71 to 103 dph. Eggs spawned on 102 and 103 

dph were collected and placed in 100 mL beakers containing 80 mL of test solution. Test 

solution was replaced daily until all embryos hatched and then 15 larvae per replicate (4 replicate 

tanks per treatment (60 larvae per treatment) with the exception of the 17.7 μg/L treatment where 

fewer embryos from the three pairs of F0 fish only allowed 2 replicate tanks) were transferred to 
the same flow through system as used for the F0 generation. F1 fish were exposed until 60 dph 

when all fish were sacrificed and sampled for secondary sex and histological analyses. 

The highest treatment (183 μg/L) resulted in 100% mortality of the F0 generation by 10 dph. 

The gonadal sex ratio (based on histological examination) of F0 fish in the 51.5 μg/L Chemical X 

treatment was significantly different from control at 60 dph. Induction of testis-ova in the 

gonads of the F0 fish at 60 dph was observed in both the 17.7 and the 51.5 μg/L Chemical X 

concentrations (Table 4). The induction of testis-ova in the gonads of 17.7 and the 51.5 μg/L 

Chemical X F0 fish at 60 dph was concurrent with significant higher mortalities (20% and 35%, 

respectively) as compared to the control of (Figure 14). 

Table 4. Total length and body weight of F0 fish at 60 d posthatch and their sex ratios as 

determined by gross examination of secondary sex characteristics and by gonadal histology. 

Adapted from Yokota et al. 2001. 

Figure 14. Post-swim-up cumulative 

mortality from 20 to 60 d posthatch in each 

Chemical X treatment (4.2 – 51.5 μg/L) of 

the F0 generation. Concentrations are 

expressed as mean measured concentrations 

(μg/L). * and ** denote significant 

differences from the pooled controls at p = 

0.031 and 0.002, respectively. Adapted 

from Yokota et al 2001. 

Because there were no phenotypic males based on secondary sex characteristics, in the 51.5 μg/L

treatment, no breeding pairs were established in the reproductive phase of the experiment (71 to 

103 dph). There were no significant effects on fecundity or fertility of F0 medaka exposed to 4.2, 

8.2 or 17.7 μg/L concentration of Chemical X. Female GSI (%) was significantly increased in 

the 8.2 and 17.7 μg/L Chemical X treatments as compared to the control at the end of the 

reproductive phase (Figure 15). 
Figure 15. Gonadosomatic index (GSI) in 

male (A) and female (B) of paired medaka (F0) 

at the end of the reproductive phase. Data 

were expressed as mean ± standard deviation. 

The sample size in each treatment was 6, 

except in the 17.7 μg/L treatment (n = 3). * 

and ** denote significant differences from the 

pooled controls at p = 0.050 and 0.002, 

respectively. Adapted from Yokota et al 2001. 

In the F1 generation, the only effect observed was a significantly different gonadal histology sex 

ratio in the 17.7 μg/L Chemical X treatment as compared to the control (Table 5).

Table 5. Cumulative mortality, growth, and sex ratios as determined by gross examination of 

secondary sex characteristics and by gonadal histology at 60 d posthatch of F1 medaka. Adapted 

from Yokota et al. 2001.

In summary, Yokota et al. (2001) found no effects of Chemical X in the F0 generation below the 

levels which produced significant mortality (17.7 μg/L Chemical X) with the exception of an 

increase in female GSI (%) at 8.2 μg/L Chemical X as compared to control. In the F1 generation, 

there was no treatment induced mortality, but there was a skewed gonadal histological sex ratio 

in the 17.7 μg/L treatment as compared to control. 

Overall, the literature reviewed in this section indicates that Chemical X has endocrine responses 

in fish consistent with an estrogen agonist at doses where there is no overt toxicity. These effects 
consist of increased VTG in males and decreased number of spawnings observed in fathead 

minnows and increased female GSI and skewed sex ratios in medaka. 

2. DISCUSSION

D. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis

i. Effects on Estrogen

Table 6 summarizes the results of the studies reviewed in this case study and endpoints relevant 

for determining the potential for Chemical X to interact with the estrogen pathway. Chemical X 

displays clear estrogenic activity in the in vitro assays examined. The chemical binds to the 

estrogen receptor and increases ER-linked gene activation. Further, data from several types of in 

vitro ER binding and ERTA assays suggest Chemical X’s interaction is both competitive and 

specific. A number of published Uterotropic assays support the estrogenic activity of Chemical 

X based on increased uterine weight. Chemical X also significantly advanced puberty (age at 

vaginal opening), altered estrous cyclicity, and reduced ovarian weight when administered to 

prepubertal female rats, further supporting hypothesized ER-mediated interaction of Chemical X

with the estrogen pathway. Chemical X also resulted in altered steroidogenesis and aromatase 

assay results. Chemical X inhibited aromatase enzyme activity in a dose-dependent manner in 

several independent studies. These data suggest Chemical X is capable of altering hormone 

production along the steroidogenic pathway, however, in vivo data support the estrogenic nature 

of Chemical X, and this effect is apparently strong enough to obscure any potential effects 

Chemical X may have on steroidogenesis. In several mammalian studies, age at vaginal opening 

was significantly advanced and uterine weight was increased, though this effect appeared to be 

most pronounced when females were exposed to Chemical X before reproductive maturity. The 

length of phases of the estrous cycle and total number of estrous cycles were altered by chemical 

exposure, though again, this effect was less pronounced and observed at higher concentrations 

when ovariectomized females were exposed to Chemical X in adulthood. Reduced gonadotropin 

levels and fecundity and fertility among mammals support hypothesized interaction of Chemical 

X with the hypothalamic-pituitary-ovarian axis.

Chemical X induced treatment-related effects on fish, though the fathead minnow and medaka 

studies reviewed in this case study were not identical to the Tier 1 EDSP Fish Short-Term 

Reproduction Assay (OCSPP 890.1350), and accordingly, more difficult to infer potential 

estrogen pathway interactions from these results. Exposure to Chemical X did increase VTG in 

male fathead minnows and reduce spawnings and total number of eggs spawned . The incidence 

of phenotypic and gonadal sex mismatch was significantly greater among treated medaka than 

controls and F1 female medaka showed a significantly increased GSI to untreated medaka. 

Based upon WoE evaluation of available data, Chemical X interacts with the estrogen signaling 

pathway and HPG axis.
Table 6. Estrogenic/Anti-Estrogenic Pathway

Lines of Evidence Indicating Potential Interaction with the 

Estrogenic/Anti-Estrogenic Pathwaya

Study Type /

Literature 

Citation

E

R

Binding

E

R

Activation

SteroidogenesisUterine WeightOvarian Weight orFish GSIOvarian/GonadStaging andHistopathologyPituitary WeightEstrous Cyclicity

b

Age &

Weight at V

O

Fertility/FecundityVitellogeninEstradiolOvert ToxicityObserved

C

EDSP Tier 1

ER Binding P

ERTA P

Aromatase P

Steroidogenesis P

Uterotrophic P

Female Rat ↑ ↓ N - P ↓ ↓ -

Fish ↑ ↑ ↓ ↑M -

a Key to responses: Positive (P), negative (N) or equivocal (E) observation; arrows (↓ or ↑) indicate the direction of the 

response; A shaded cell indicates that parameter was not evaluated or is not applicable.

b Factors for estrous cyclicity include: age of first estrous, length of estrous, and percent of animals cycling regularly.

c An “X” in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity. 

ii. Effects on Androgen Pathway

Table 7 below summarizes the results of the data reviewed in this case study and endpoints 

relevant for determining the potential for Chemical X to interact with the androgen pathway. 

Various targets of the androgen pathway are delineated so as to facilitate determination of 

potential for androgenic, anti-androgenic, or HPG axis effects. 

Chemical X appears to interact with the androgen receptor in several in vitro assays, though 

interactions appear to vary somewhat with the receptor test system used. Chemical X represent a 

number of isomers that likely vary in their affinity for hormone receptors. The difference in 

isomers evaluated in different studies may explain disparate results. Chemical X significantly 

reduced binding of physiological ligand to the rat AR. Several AR transcription assays indicated 

an antagonistic effect of Chemical X on reporter gene expression. As discussed for the estrogen 

pathway, Chemical X may interfere with steroidogenesis, based on significant inhibition of 

aromatase enzyme activity, reduction in steriodogenic enzyme transcripts other than aromatase,

and a biphasic alteration in testosterone production observed in in vitro assay described in 

pervious sections. 

The generally antagonistic interactions of Chemical X with the androgen pathway observed in in 

vitro assays were not uniformly supported by in vivo study data. Though an EPA interlab 

validation study did support limited antagonistic action of Chemical X on androgen-responsive 

tissue weights in the Hershberger assay, none of data from the 10 labs involved in the interlab 

trial using Chemical X produced data that met the EDSP guideline criterion for androgen 

antagonism. In other Hershberger studies, Chemical X is clearly negative for both agonist and 
antagonist activities, and in fact, Chemical X is sometimes used as a negative control in this 

assay. 

Effects of Chemical X treatment on the rat androgen signaling pathway appear to vary with age 

of exposure. Animals exposed in the postnatal or pre pubertal stage had significant alterations to 

androgen levels and/or gonadal histology. Animals exposed after puberty appeared to be less 

effected, though testosterone was significantly increased along with changes to the thyroid 

hormone pathway, suggesting these effects may be mediated through the HPG/HPT axis.

Early postnatal and prepubertal exposure resulted in reduced copulatory behaviors and germ 

cells, testis weight, and in some instances increased testosterone concentrations concomitant with 

elevated LH. When exposed at 6 weeks of age, Chemical X resulted in reduced testis weight but 

no other changes to male reproductive parameters. Additionally, there appeared to be no effect 

of Chemical X in a multigenerational rat study. 

It is important to note that this case study reviews OSRI rather than EDSP Tier 1 studies 

validated to screen for the potential of chemicals to interact in the EAT pathways. While many

studies reviewed are similar in underlying scientific principles, they are not identical in execution 

and thus, sensitivity. Many results summarized in this case study may be the result of alterations 

in the estrogen pathway or androgen pathway. In validation studies, treatment with 17β estradiol 

or ethinyl estradiol results in similar perturbation. Chemical X appears to interact with the AR 

and inhibit receptor-mediated transcription, however, the absence of significant results in the 

Hershberger assay, considered diagnostic for the A pathway, calls into question the ability of 

Chemical X to interact in the androgen pathway in in vivo test systems. Based upon WoE 

evaluation of available data, Chemical X may alter androgen signaling pathway and the HPG 

axis.

Table 7. Androgenic/Anti-Androgenic Pathway

Lines of Evidence Indicating Potential Interaction with the Androgenic/AntiAndrogenic Pathwaya

Study Type /

Literature Citation

A

R

Binding

SteroidogenesisTestosteroneTestes Weight orMale Fish GSIGonad Staging andHistopathologyEpididymidesWeightEpididymidesHistopathologyPituitary WeightAccessory SexOrgan Weights/2°Sex CharacteristicsAge and Weight atP

PS

Fecundity/fertilityVitellogeninOvert ToxicityObserved

b

EDSP Tier 1

AR Binding P

Aromatase P

Steroidogenesis P

Hershberger -

Male Rat ↓ ↓ ↑ ↓ ↑ ↑ - -

a Key to responses: Positive (P), negative (N) or equivocal (E) observation; arrows (↓ or ↑) indicate the direction of the 

response; A shaded cell indicates that parameter was not evaluated or is not applicable; L, M, and H refer to low, middle and 

high test concentration, respectively; ♂ = male; ♀ = female

b 125 mg/kg bw/day dose in supplemental chronic/carcinogenicity study

E Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis
i. Thyroid Hormones 

Table 8 below summarizes the results of the data reviewed in this case study and endpoints 

relevant for determining the potential for Chemical X to interact with thyroid regulation. The 

various targets of the thyroid pathway are delineated so as to facilitate determination of potential 

for thyroid or HPT axis effects.

The EDSP Tier 1 battery includes male and female pubertal rat studies with thyroid endpoints 

and an amphibian metamorphosis assay (OCSPP 890.1100) for screening chemicals with the 

potential to interact with the thyroid pathway. While Chemical X does not have any Tier 1 

screening submissions, several studies discussed in the case review included endpoints that may

provide anecdotal information about the potential to interact with the thyroid pathway. No 

equivalent EDSP Tier 1 assays designed to evaluate potential interaction with T were available 

for this case study.

Nagao et al. (2000) reported increased thyroid and pituitary weights and altered plasma T3, T4

and TSH. When treated over two generations, TSH was significantly increased, along with 

thyroid gland weight, in adults F0 males exposed to the highest dose (50 mg/kg/d) of Chemical 

X, and significantly decreased in F1 PND 22 males and females exposed to low (2 mg/kg/d) and 

high doses. T3 and T4 were significantly decreased in F0 adults treated with low concentrations 

of Chemical X, and concentrations remained low in males exposed to high dose, though T3 of 

females exposed to the same concentration was elevated. In the absence of a clear-dose response 

or consistent relationship, these results are difficult to interpret. It is worth noting that Chemical 

X displays clear and somewhat consistently non-linear dose-response effects in the E and A 

pathways, so the thyroid effects summarized in this case study may be a result of exposure to the 

chemical. 

Interactions with the T pathway are not clearly delineated by the data summarized in this case 

study, however the studies reviewed not include assays specifically designed to detect potential 

chemical interaction with the thyroid. Because of the limited data and ambiguous results, the 

potential of Chemical X to interact with the thyroid hormone pathway cannot be excluded.
Table 8. Thyroid Pathway

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathwaya

Study Type /

Literature Citation

Hormones (T4 and TSH)Pituitary WeightThyroid WeightThyroid Gross andHistopathologyFrog Development StageHind Limb LengthSnout to Vent LengthOvert ToxicityObserved

b

EDSP Tier 1 

Male Rat ↑ (Fo adult) 

↓ (F1 juv) ↑ ↑M altered

Female Rat ↑ (F1 juv)

T3

- - -

a Key to responses: Positive (P), negative (N); arrows (↓ or ↑) indicate the direction of the response; A shaded cell indicates 

that parameter was not evaluated or is not applicable.

b An “X” in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt 

toxicity

c While overt toxicity was observed in this assay in the two of the three treatment groups, at the lowest treatment group, 

where there was an no overt toxicity observed, there were no indications that the effects observed (delayed development and 

growth) were due the chemical’s interaction with the thyroid pathway.

3. CONCLUSIONS

F. Summary of Potential Endocrine Effects

Based on the studies evaluated in this WoE evaluation of Chemical X, there is strong support 

from in vitro and in vivo data that Chemical X has the potential to interact with the estrogen 

pathway. It is important to recognize that “Chemical X” is in fact a collection of several isomers 

and composition of isomers is not always reported. The degree of side chain branching may

influence the affinity of Chemical X for the ER, and perhaps other potential endocrine 

interactions. Chemical X appears to inhibit aromatase enzyme activity and may alter transcripts 

of steriodogenic enzymes upstream to estrogen synthesis. In vitro AR binding assays and 

androgen receptor transactivation assays suggest Chemical X may be capable of interacting with 

the AR in a generally antagonistic manner. These data were not corroborated by results of the 

Hershberger assay, though rat and fish in vivo assays suggest that Chemical X may act as an 

estrogen or antiandrogen in male animals and appears to be capable of interacting with the HPG 

axis. Research summarized in this case study did not include experiments designed specifically

to test for interaction in the thyroid endocrine pathway, though limited data suggest potential 

interaction with the mammalian thyroid system. Though results discussed in this evaluation

must be regarded carefully and do not indicate clear relationships, the potential of Chemical X 

with the thyroid hormone pathway cannot be discounted.
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