### EDSP: W EI GHT OF 

### E VI DENCE 

### A NALYSIS OF 

### T IER 

### 1 S TUDIES



### C ASE 

### S TUDY 

### : C HEMICAL 

### X

O FFICE OF PESTICIDE P ROGRAMS

OFFICE OF SCIENCE CO ORDINAT ION AND POLIC Y 

### O FFICE OF 

### P OLLUT IO N 

### P REVENTION AND 

### T OXICS 

### U.S E NVIRONMENTAL 

### P ROTECTION 

### A GENCY**1. Case Study** 

**– Chemical X** 

**A**
**. Introduction** 
Chem ical X is presented as a specific case study to illustrate the process of apply ing a Weight of Evidence ( WoE ) anal ys is to determine a chemical’s potential to interact with the endocrine sy stem. Unlike the EDSP L ist 1 case studies, Chemical X was not subject to EDSP Tier 1 screening and, therefore, only Other Scientificall y Relevant Information (OSRI ) is available for consideration in th e 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 s ys tem. Chemical X is a non

- pesticide that comes from a family of closely

related, commerciall y important alky lphenols used in the production of ethoxy lates which have a wide variet y of industrial applications and are produced in large volumes. The endocrine activity of Chemical X and its associated ethox ylates and carbox yl ates has been well studied in vitro and in vivo and, therefore, was chosen as a case stud y to illustrate the WoE process solel y based on OSRI.

The OSRI ch osen to represent the potential for Chemical X to interact with the endocrine s ys tem consists primarily

of published, scientifi cally 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 assay s. It is emphasized that this case study , as with the EDS P L ist 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 anony

mous 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 st anding of Chemical X . The OSRI selected and the WoE analy ses presented for this case should not be interpreted as comprehensive, final or complete bey ond this limited illustration. The agency will fully evaluate Chemical X , taking into account SAP commen ts o n the WoE process , to determine whether or not Chemical X has the potential to interact with the endocrine s ys tem when the final SAP report is available. The SAP is asked to include Chemical X along with the other chemicals in their responses to respe ctive charge questions.
**B**
**. Data Available for**  **Chemical X** Chemical X is a phenolic compound with multiple isomers usually found as a pale y ellow, highl y viscous liquid, with an approximate molecular weight of 215.0 to 220.4 g/mole, specific gravity of 0.95 3 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). I t has a dissociation constant (pK a ) of 10.7 ± 1.0 and a log octanol/water partition coefficient (log K ow ) of 3.80 to 4.77 (Roy F. Weston I nc. 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 so lvents (Roy

F. Weston I nc. 1990). Ahel and Giger (1993) measured the solubility of Chemical X at different temperatures in distilled water and demonstrated a nearl y linear increase in solubility between 2°C (4,600 μg/L) and 25°C (6,350 μg/L). The c hemical has and does not volatize appreciabl y and is characterized as being moderatel y persistent , as it isresistant (stable) to abiotic routes of degradation ( i.e., hy droly sis and photoly sis) and to

anaerobic biotic metabolism. The relativel y high log 10 octanol

- water partition coefficient and high organic carbon partition coefficient (K oc ~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 lipophil ic, it does not bioaccumulate appreciabl y in fish (BCF range 90

– 330x).

**C. Other Scientifically Relevant Information (OSRI)**  **for Chemical X**
In formation that is submitted voluntarily and is applicable to support or clarify an EPA action is generall y referr ed to as “other” scientifically relevant information. Sources of relevant scientific and technical information may include toxicity

study results from EPA or OECD eq uivalent test guideline s and information from published or publically available peer

- revie wed studies. Regardless of the source, the information is evaluated for qualit y and relevance, in accord with EPA’s guidance on scientific integrit y ( 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 L ist 1 case studies for continuity

. T he results from OSRI for the various modes or pathway s of action involving E, A and T for receptor binding (agoni s t and antagonist) and a ctivation/transcription, steriodogenesis (competitive induction or inhibition) , and the hy

pothalamic

- pituit a ry - gonadal and

- thy roidal axes are summarized in the following sections in context with respective EDSP Tier 1 screening assay s. 

### i. **ER Binding Assay s**  The results from s everal studies which are technically similar to the EDSP Tier 1 ER binding assay (OCS PP 890.1250 ) that have demonstrated the potential of Chemical X to bi nd with the estrogen receptor are presented. In a study by Danzo ( 1997) , ER bindi ng was observed using rabbit uterine cy

tosol incubated with 7 nM [ 3 H] 17 β

- estradiol alone overnight at 4 o C 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 ( 3 H)estradiol ( E2 ) to the estrogen receptor a s shown in **Figure 1**

.**Figure 1.** The mean ± standard error is shown. Percent inhibition of 3 H estradiol binding to the

estrogen receptor is determined for a variet y of chemicals. On the far left, the specific binding of 3 H estradiol (*E) is shown (100%). Second from the left, unlabeled estradiol (E) results in 100% inhibition (i.e. 0% [ 3 H] 17β

- estradiol bound to receptor). Chemical X ( 10 0 μM ) is indicated by the red box. The numbers in parentheses above the bars indicate the number of experiments in which sam ples were assay ed in triplicate

. Figure ad a pted 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 IC 50 of 2.4 to 4.7 x 10 - 6 and a RBA of from 0.02 to 0.037 % as shown in **Figure 2**
**. Figure 2.**  Demonstrated a ffinity of Chemical X ( open triangles) for the ER appears to be parallel to the linear portion of the curve of natural ligand estr adiol ( closed circles ). Figure ada pted from Blair et al., 2000.
In an investigation by Satoh et al. ( 2001) , human ER α binding was quanti fied using fluorescence

- labeled 17β

- estradiol , incubated with Chemical X for 2 hr at room temperature , Chemical X bound to ER α with an IC 50 of 7.0 x 10 - 7 M and an RBA of 2.22%.
Laws et al. (2000), i n a large study includi ng multiple in vivo experiments substantiated by in vitro ER binding and transcription activation, reported weak affinit y of Chemical X for the rat uterine cy tosolic ER as demonstrated by K i of 0.67 μM compared to 0.4 nM for 17

- β

- estradiol

. Chemical X represents a variet y 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 r esults of estrogen rece ptor binding assay s conducted in different cell backgrounds and following different methods indicate Chemical X bind s to the estrogen receptor under a variet y of assays conditions. Thus, b ased on the combined results , Chemical X is considered a positive fo r ER binding in competitive receptor binding assay s
.**ii. ER Transcriptional Activation Assays** 

Balaguer et al (1999) utilized four reporter gene assay s 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 ER α expression, transfected with an ER

- responsive luciferase reporter. The authors also used a HeLa cell line containing endogenou sl y expressed ER stably transfected with ER α , ER β , or a chimeric ER α receptor along with a luciferase reporter gene. After 3 day s 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

.

**Fig ure 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 assay s. 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 E 2 10 - 8 M (9500 arbitrary units/0.1 mg of protein ). Estradiol (E 2 ) 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 MVL N cell line, which are MCF

- 7 cells stab ly transfected with a luciferase reporter. Chemical X was tested in three independent runs for agonist and antagonist activit y at concentrations ranging from 10 - 8 to 10 - 4 M. Chemical X produced a clear estrogenic response with an EC 50 of 8.9 μM. No antago nist activity

was observed when Chemical X was tested in the presence of 25 uM.**Figure 4**
**.** D ose

– response ER transactivation of E 2 and Chemical X. The graph on the left shows relative luminescence in MVL N cells exposed to E 2 (0.05

– 500 pM, closed circles) and to 10 - 8 to 10 - 4 M 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 E 2 , which was set to 1. Mean values are shown (n ≥ 3). *Significantl y different from the respective solvent controls (cells + 0.1% DMSO; 25 pM E2 + 0.1% DMSO). Adapted from Bonefeld

Jorgenssen et al . 2007.
U sing an ER

- mediated, chemical

- activated luciferase reporter gene (ER

- CAL UX) expressed in human T47D breast cancer cells , Legler et al . (1999) determined that Chemical X was among the most potent chemicals tested with an EC 50 of 260 nM.
White et al. (1994) tested the effect of 4

- Chemical X in rainbow trout ( Oncorhynchus mykiss ) hepatocy tes and several human breast cancer cell lines. Chemical X increased vitellogenin gene expression in trout hepatocy tes 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 - 6 to 10 - 5 M.**Figure 5**
**.** ER

- mediated gene induction in A) rainbow trout hepatocy tes and B) MCF

- 7 human breast cancer cells. The graph on the left shows vitellogenin induction in rainbow trout hepatocy tes 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 - 8 E 2 is indicated by the first bar on the graph. Adapted from White et al . 1994.
Tho ugh different reporter gene constructs and cell backgrounds may have different sensitivities to native ligand and Chemical X, a variety of ER transcription activation assay s comparable to the Tier 1 ERTA or quantify ing similar ER

- mediated gene activation s upport 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 assay s
. **iii. AR Binding Assay**  **s** The potential of Che mical X to bind the androgen receptor was tested in a variet y of assay sy stems

. Satoh et al. (2001) incubated human androgen receptor for 1 hr incubation at 4 o C with unlabeled testosterone, followed by the addition of an anti

- testosterone antibody and peroxidase

labeled 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 I C 50 and relative binding affinit y 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. L ig and binding experiments were carried out using a Ligand Screening S ys tem

- Androgen Receptor kit (To yobo, 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 ye ast transformed with full length mammalian AR and a luciferase reporter plasmid conta ining two AREs and a prostate specific antigen promoter, Lee et al. (2003) reported 5 nM Chemical X treatment inhibited [3H]5  - dihy drotestosterone (DHT) binding to the AR by 30% in HeLa cells transfected with a luciferase

- linked mouse AR, though the respon se was not dose

dependent . Further, in transient transfection assays, Chemical X inhibited AR transcriptional activity (I C 50 = 2.6 μM) and was a more potent inhibitor of androgen

- induced gene expression than recognized antagonist cy proterone 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 IC 50 of 14.1 μM, and a maximum inhibition (MI ) of 56 %. Though high concentrations of Chemical X can cause cy totoxicity , the authors report ye ast cell growth (measured b y absorbance at 600 nm) was not significantly reduced in the concentrations of Chemical X t ested in this study

. Chemical X has been shown to significantl y reduce AR gene transcription in Chinese hamster ( Cricetulus griseus ) ovary cells transfected with human AR and a luciferase reporter (Bonefeld

Jorgensen et al ., 2007). The potential AR anta gonism of several compounds was tested in cells treated with increasing concentrations of Chemical X (0.15 to 40 x 10 - 6 M) in the presence of an AR agonist (0.1 nM R1881). AR binding was analy zed using a four parameter logistic curve fit and an IC 5 0 was ca lculated. Chemical X elicited an antagonist response with an I C 50 of 14.1 μM and a maximum inhibition of 56% at 10

- 6 M. Chemical X was determined to be cy totoxic to CHO cells at concentrations > 40 x 10 - 6 M.

**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

assay s, though it is important to note that steroidal estrogens and xenoestrogens will elicit a positive response in AR binding assay s. **iv**
**. Steroidogenesis Assays**  Effects of Chemical X on steroidogenesis were examined in assay s ys tems substanti ally

different than the in vitro EDSP Tier 1 Steroidogenesis assay (OCSPP 890.1550). L ey dig 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% dimethy lsulfoxide ( DMSO ) . Cell viability was tested by try pan blue exclusion. Testosterone production measured after 48 hr was significantl y increased in the low concentrations and reduced at highe r concentrations. C oncentrations 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 Le ydi g cell death at high concentrations.

**Figure 8**
**.** Testosterone concentrations in media of L ey dig 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 enzy me mRNA quantified by

real

- time PCR in in vit ro Atlantic salmon ( Salmo salar ) ovarian tissues exposed for 3 and 7 day s
. Expression of steriodogenic acute regulatory protein (StAR), side

- chain cleavage enzy me (P450scc), 3 β

- hy droxy steroid dehy drogenase (3 β

- HSD), and 17α

- hy drox yl ase/17, 20 ly ase (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 expos ure concentration. Tissue levels of ovarian testosterone and 11

- k etotestosterone were significantly elevated following 7 day s 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 stud y (RTI I nternational, 2007) during the inter

laboratory

validation studies for the Tier 1 Aromatase assay guideline (O SCPP 890.1 200). Briefl y, Chemical X was incubated for 15 min with human re combinant aromatase and tritiated androstenedione ( 1

- β [ 3 H(N)]

- a ndrost

- 4

- ene - 3,17

- dione ( [ 3 H]ASDN)) dis s olved in ethanol and a romatase 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 μ M) including a known inhibitor ( aminoglutethimide ), and a range of concentrations of the test chemical (5, 7.5, 10, 15, and 20 μ M) with 2 replicates per concentration. Chemical X inhibited aromatase activity with an IC50 of 21 μ M

. In a second stud y, aromatase activity was measured in a human choriocarcinoma cell line was measured b y tritiated water production from 3 H

- androstenedione substrate. Chemical X was added to cells and incubated for 18 hr after which cells were washed and provided with 0.2 μCi [1β

3 H] 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 anal yz ed for tritiated water production as a measure of aromatase activity

. All concentrations of Chemical X were tested in triplicate and run in three independent assay s, each of which included a p ositive control, the known aromatase inhibitor, 4

- androsten

- 4

- ol

- 3,17

- dione. Chemical X concentrations in the range of 10 - 9 to 10 - 5 M resulted in a maximum inhibition of 71% observed at 10 - 5 M (Bonefeld

Jorgensen et al. 2007).
Based on the results of these studies, Chemical X appears to be an in vitro inhibitor of aromatase.
**vi**
**. Uterotrophic Assay**  **s** The re sults from several studies done in a u terotrophic assay s similar in design and methodology to the EDSP Tier 1 screening assay ( OCS PP 890.160 0 ) 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 desig ned to evaluate the effects of C hem ical X on uterine weight in sexually immature females rats (prepubertal) at 6 and 24 hours following the last of 3 daily dose s (25, 50, 100, and 200 mg/kg) when administered b y oral g avage for 3 day s starting on PND 21

- 24

. P ositive controls were 17 β

- estr adiol (E 2 ) and e thy ny l estradiol (EE)

. W et uterine weig ht ( i.e. , uterus and fluid in uterine lumen) expressed as perce nt 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 similarl y designed experiment from the same stud y (Laws et al., 2000), the effects of Chemical X on uterine weight when administered by subcutaneous injection or oral gavage were examined. Both dosing rout es resulted in an increase in uterine weight , al though 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 ( E 2 ) by

sc injection, or to eth yn yl estradiol (EE ) , or C hemical 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 b y Dunnett’s Multiple Comparison Test, Mann

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

.

**Figu r**  **e 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) b y sc injectio n, or to ethy ny l estradiol (EE; second data set from left), or chemical X (red box) by oral gavage. *Significant treatment effect b y ANOVA or the Kruskal

- Wallis Nonparametric Anova Test ( p = 0.05) with comparison to the control byDunnett’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 stud y by L aws et al. (2000), the effect of Chemical X on uterine weight in intact prepuberal and adult ovariectomized fe male rats was compared. Uterine weight s in adult ovariectomized females w ere significantly increased as shown in **Figure 11**  , but at doses greater (100 mg/kg) than required to increase uterine weight in the pre pubertal rats as shown in **Figure s**  **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 ethy ny l estradiol (EE; second data set from left), or chemical X (red box) by oral gavage. *Significant treatment effect b y 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 , 200 3 ). Briefl y, Chemical X was administered to adult ovariectomized rats at 15, 75, 125, 250 and 350 mg/kg/d via oral gavage for 3 consecutive day s
. A vehicle control group (corn oil) and positive control s (17 α - Ethiny l Estradiol, EE) were included to evaluate estrogen agonist activity

. A ll animals survived to the end of treatment in the 15, 75 and 125 mg/kg dose

- groups; however, extensive mortality in the 2 50 and 350 mg/kg dose groups ( 38 and 67% , respectivel y ) indicated overt toxic ity at these tre atment levels

. At lower doses, u terine weight was statistically increased in the 75 and 125 mg/kg/d treatment groups in all 4 participating laboratories. Thus, based on the combined r esults of the Uterotrophic A ssay s similar in design and methodology to the EDSP Tier 1 screening assay , Chemical X is positive for estrogenic activity as indicated b y a significant increas e in uterine weight.**vii**
**. Hershberger**  **Assay s** 

In a Hershberger assay that was designed similar to the EDSP Tier 1 screening assay (OCS PP 890.1400), Frey

berger 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 suspensio n orally and subcutaneousl y for 10 day s. 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 te st guideline , a statistical inc rease (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 stud y
. 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 ). Briefl y, Chemical X was administered at 160 mg/kg/d via oral gavage using the castrated rat model. A ve hicle control group as well as positive control groups to evaluate androgen agonist ( TP) and antagonist (FLU) activities were included. Pooled across multiple l aboratories, 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 s tatistically

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 tar get tissue weights compared to the control. For the other 5 laboratories, a statistically significant decrease was detected in one or more target tissue s. Notably , for those laboratories in which only one target tissue was affected, the tissue was different in each of the 4 laboratories. In o nly 1 of the 5 laborator ies was a significant decrease detected in two of the target tissue s that would be indicative of a positive response. Thus, based on the combined results of the Hershberger Assay s similar in design and methodology

to the EDSP Tier 1 screening assay , androgenic activity of Chemical X was n egative.

**viii**
**. Pubertal Female**  **Assay**
The results for the pubertal female assay s 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) p repubertal female Sprague

- Dawley rats were dosed for 20 day s beginning on PND 21 to 10, 50, and 100 mg/kg of Chemical X delivered by oral gavage. Estrogenic activit y of Chemical X was indicated by a significant advance in age at vaginal opening (VO) . Irregular estrous cy clicit y was characterize d by increases in time to diestrusfollowing exposure to Chemical X at 50 and 100 mg/kg/d treatment

. Ovarian weight was

significantl y decreased among female s exposed to the high dose (100 mg/kg/d)

. Th yr oid effects were indicated b y a dose

- dependent d ecrease in serum T4

. In a study b y 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 pubert y
. All treated females experience d irregular estrous cy cles with shortened diestrus

. In creased atretic follicles and decreased corpora lutea were observed in ovaries and increased incidence of uterine histopathology

wa s observed among treated females. Copulation and fertilization rates among male:female pairs were significantly reduced when compare d to controls , as were the number of live embry os born to dams treated with Chemical X in the early postnatal period. In a study b y 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. A ge at vaginal opening was significantl y advanced ( i.e. , the age of animals at VO decreased) following treatments of 50 mg/kg and greater . The number of estrous cy cles was significantly decreased following 100 mg/kg Chemical X dosing as shown in **Table 1** , al though the same dose failed to alter vaginal cy tology of ovariectomized females following 11 day s of exposure and only affected estr us cy clicity of adult, intact females following longer exposures ( 25 day s ) of 100 mg/kg of Chemical X administrated orall y
. **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 F 0 (parent) generation were treated from two weeks prior to pairing until necrops y (21 day s after delivery of th e F 1 generation). First generation ( F 1 ) females were dosed indirectly through lactation and directl y from weaning (PND 21) until necropsy (21 day s after delivery of the F 2 generation) and the F 2 generation was terminated at weaning (PND 21)

. Chemical X do sing was continuous from the initiation of F 0 generation through necrops y of the F 2 animals. Neonatal s urvival (i.e., n umber of live F 1 and F 2 pups per litter) was significantly re duced in the high treatment group (50 mg/kg) , although postnatal growth was not affected. T reatment did not significantly affect estrous cy cle s or result in histopathological lesion s in any reproductive tissue s examined. Absolute and relative uterine weight s were significantly increased among fema les exposed to 2 mg/kg and significantl y decreased in the 50 mg/kg group

. Ovarian weight was significantly decreased and v aginal opening was significantly advanced in the high treatment group, al though treatment did not have an effect on estrous cy cle s , m ating, or fertility

. On postnatal day 22, serum concentrations of L H 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 v ia the diet. C alculated dose rates for yo ung 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 respectivel y. Adult and PND 21 animals were necropsied. Vaginal opening was advanced ( i.e. , age decreased) in all genera tions and uterine weights were increased in the mid and high dose groups for onl y 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 signi ficant changes in various estrogen

depende nt endpoints. The results on thy roid hormones was inconsistent

. **ix**
**. Pubertal**  **Male As say**  The results for the pubertal male assay s 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 stud y 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 delay ed pubertal onset, increased incidence of histopathology observed in the testis, and affected spermatogenes is 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 s ubcutaneousl y and examined animals through puberty

. No sign ificant alterations to behavior or plasma testosterone were observed and the increased time to preputial separation was not significant ly altered from control

. Chemical X

- treated rat body weight was significantl y reduced in males (though reductions were < 10% terminal body weight), however, relative testis we ig ht was also significantl y reduced. Chemical X exposure may have altered male fertility

as indicated by gonad al histopathological examination. There was a d ecrease in germ cells in the seminiferous tubules and increased degenerate germ cells in the e pid idy mides

.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 we eks prior to pairing. No adverse effect s of Chemic al 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, al tho ugh the absolute weight of the e pidid ym ides of the 2 mg/kg treatment group were significantly greater than controls. Relative thy roid and pituitary

gland weights were elevated among the high treatment groups. Preputial separation and anogenita l distance were not affected. No histopathologi cal lesions were observed in reproductive tissues ; however, there was a significant increase in testosterone . Histologicall y, the th yroid gland appeared enlarged in the 50 mg/kg treatment group, and t here was a dose d ependent increase in TSH and a signifi cant decrease in T 3 a nd T 4 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 day s with 125 and 250 mg/kg/d of Chemical X administered b y oral gavage for 50 day s and measured serum testosterone and luteinizing hormone

. . Terminal samples indicated testosterone lev els had dramatically declined in the high dose group , while LH increased in the 125 and 250 mg/kg/d dose groups. DeJager et al. (1999 a ) reported on the effects of Chemical X on male fertility after direct exposure to adult Sprague

- Dawley rats in an OECD One

- G eneration R eproductive Toxicity S tudy (OECD Test Guideline 415, 1983 ). A dult 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 bey ond the duration of normal spermatogenesis and epididy mal transit time to cover at least one complete spermatogenic cy cle ( i.e., approximately 52 day s)

. Endpoints included body mass, testicular and epididy mal mass, cauda epididy

mal sperm count, and testicular histology

. A dose

- dependent decrease in s urvival 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 t estis mass:body mass ratio was significantl y reduced (36 and 18%, respectivel y) in the high

- dose group compared to the control group. Epididy mal mass and ratio was significantly reduced in the mid

- (12 and 10%, respectively ) and high

- dose (56 and 44%) grou ps compared to controls. Total caudal epididymal sperm count was significantl y reduced (79%) in the high

- dose gro up compared to controls

. Testicular histology indicated normal spermatogenesis encompassing all 14 stages in all treatment groups ; however, s eminiferous tubule diameter s were significantl y 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, respectivel y, 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 epididy mis and number of sperm.**Table 2**
**.** Adapted from DeJag er et al., 1999a.

In an o ther stud y, 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 or al gavage for 10 weeks to cover at least one complete spermatogenic cy cle (i.e., approximately 52 day s). Endpoints included body mass, testicular and epididy mal mass, cauda epididy

mal 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. Bod y mass , represented b y the difference in bod y mass from weanin g 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 low

a nd mid

- dose group compared to the control group. Epididy mal mass was significantl y reduced in the mid

- dose groups compared to controls. Overall anal ys is of the epididy mal ratio was not significantl y different among groups. Total caudal epididy mal 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 m id

- 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 mid

dose 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 overtl y toxic at 20% in the mid

- dose group. Nonetheless, the negative impact on testicular and epidid ym al 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

- a ndrogenic in the adult male rat . Though man y endpoints altered b y Chemical X in the studies described in this section are not specific to either the E or A pathway, results strongl y suggest the potential of chemical X to interact in the male rat endocrine s ys tem.**x. Fish**  **Endocrine Relevant Studies**
Harries et al . (2000) performed a short

term (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 b y 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 volum e with a flow

through 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 12**  **A** ) for the exposure period, but there were no significant differences between breeding pairs in the pre

- exposure period. The mean number of spawn ings 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 12**  **B** ). The highest dose of Chemical X also produced a significant reduction in mean egg batch s ize per breeding pair ( **Figure 12 C**  ). **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 control s (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 ( P imephales 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 breedi ng 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 wate r (control) in VTG levels but there were no significant differences in gonadosomatic index ( **Fi gure**  **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 i n 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 sp awned and mean egg batch size.
Yokota et al. (2001) performed a reproduction study over two generations (F 0 and F 1 ) 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 (F 0 ) was from 24 hours post fertilization (hpf) to 104 day s post hatch (dph) with a flow through sy stem providing a 14 volume/day renewal rate to 1.8 L replicate test chambers. There were 4 repl icate test chambers from initiation (24 hpf) until 70 dph and each replicate began with 15 embry os. At 60 dph, 5 larvae per replicate were sampled for secondary sex and histological anal ys es. At 70 dph, 6 phenoty pic male:female mating pairs of F 0 fish we re 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 embry os 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 embry os from the three pairs of F 0 fish onl y allowed 2 replicate tanks) were transferred tothe same flow through s ystem as used for the F 0 generation. F 1 fish were exposed until 60 dph

when all fish were sacrificed and sampled for secondary sex and histological analy ses. The highest treatment (183 μg/L) resulted in 100% mortality of the F 0 generation by 10 dph. The gonadal sex ratio (based on histological examination) of F 0 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 F 0 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 F 0 fish at 60 dph was concurrent with significant higher mortalities (20% and 35%, respectivel y) as compared to the control of ( **Figure 14**  ).
**Table 4.** Total length and body weight of F 0 fish at 60 d posthatch and their sex ratios as determined b y 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 F 0 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 phenoty pi c 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 F 0 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 significa nt differences from the pooled controls at p = 0.050 and 0.002, respectivel y. Adapted from Yokota et al 2001.

In the F 1 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 b y gross examination of secondary sex characteristics and by gonadal histology

at 60 d posthatch of F 1 medaka. Adapted from Yokota et al

. 2001.

In summary , Yokota et al. (2001) found no effects of Chemical X in the F 0 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 compar ed to control. I n the F 1 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 effectsconsist 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 stud y and endpoints relevant for determining the potential for Chemical X to interact with the estrogen pathway

. Chemical X displ ay s clear estrogenic activity in the in vitro assay s examined. The chemical binds to the estrogen receptor and increase s ER

- linked gene activation . Further, data from several ty pes of in vitro ER binding and ERTA assay s suggest Chemical X ’s interaction i s 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 significantl y advanced pubert y (age at vaginal opening), altered estrous cy clicit y, and reduced ovarian weight when administered to prepubertal female rats, further supporting hy pothesized 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 enzy me activity in a dose

- dependent manner in several independent studies. These data suggest Chemical X is capable of altering hormone prod uction along the steroidogenic pathway , however, i n vivo data support the estrogeni c nature of Chemical X , and this effect is apparentl y strong enough to obscure an y 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 cy cle and total number of estrous cy cles were altered b y chemical exposure, though again, this effec t 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 hy pothesized interaction of Chemical X with the hy

pothalamic

- 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 accordingl y, more difficult to infer potential estrogen pat h way 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 phenot ypic and g onadal sex mismatch was significantly greater among treated medaka than controls and F 1 female medaka showed a significantly increased GSI to untreated medaka

. Based upon WoE evaluation of available data, Chemical X interacts with the estrogen sign aling pathway and HPG axis.**Table 6** 
**.** Estrogenic/Anti

- Estrogenic Pathway **Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti** 

**- Estrogenic Pathway**  **a**

**Study Type / Literature**  **Citation E** **R B** **i n** **d i** **n g** **E R** **A c** **t i** **v a** **t i** **o n** **S t** **e r** **o i** **d o** **g e** **n e** **s i** **s U** **t e** **r i** **n e** **W e** **i g** **h t** **O v** **a r** **i a** **n W** **e i** **g h** **t o** **r F** **i s** **h G** **S I** **O v** **a r** **i a** **n /** **G o** **n a** **d S** **t a** **g i** **n g** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y P** **i t** **u i** **t a** **r y** **W e** **i g** **h t** **E s** **t r** **o u** **s C** **y c** **l i** **c i** **t y** **b A** **g e** **& W** **e i** **g h** **t a** **t V** **O F** **e r** **t i** **l i** **t y** **/ F** **e c** **u n** **d i** **t y** **V i** **t e** **l l** **o g** **e n** **i n** **E s** **t r** **a d** **i o** **l O** **v e** **r t** **T o** **x i** **c i** **t y** **O b** **s e** **r v** **e d** **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 determ ining the potential for Chemical X to interact w ith the androgen pathway

. V arious 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 assay s, though interactions appear to vary somewhat with the receptor test sy stem used. Chemical X represent a number of isomers that likely vary in their affinity for hormone receptors. The differe nce in isomers evaluated in different studies may explain disparate results. Chemical X significantl y reduced binding of ph ys iological ligand to the rat AR

. Several AR transcription assay s 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 enzy

me activity , reduction in steriodogenic enzy me transcripts other than aromatase, and a biph asic alteration in testosterone production observed in in vitro assay described in pervious sections.
T he generall y antagonistic interactions of Chemical X with the androgen pathway observed in in vitro assay s 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 produc ed data that met the EDSP guideline criterion for androgen antagonism. I n other Hershberger studies, Chemical X is clearl y negative for both agonist andantagonist 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 a fter pubert y appeared to be less effected, though testosterone was significantl y increased along with changes to the thy roid hormone pathway , suggesting these effects may be mediated through the HPG/HPT axis. Earl y postnatal and prepubertal exposure result ed 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 t o 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 pathway s. W hile many studies reviewed are similar in underl yi ng scientific principles, they are not identical in execution and thus, sensitivity

. Many results summarized in this case study may be the result of alterati ons in the estrogen pathway

or androgen pathway . In validation studies, treatment with 17 β estradiol or ethiny l estradiol results in similar perturbation. Chemical X appears to interact with the AR and inhibit receptor

- mediated transcription, however, th e 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 sy stems. Based upon WoE evaluation of available data, Che mical X may alter androgen sign aling pathway and the HPG axis.
**Table 7** 

. Androgenic/Anti

- Androgenic Pathway

**Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti**

  ** Androgenic Pathway** **a**

**Study Type / Literature Citation** **A R** **B i** **n d** **i n** **g S** **t e** **r o** **i d** **o g** **e n** **e s** **i s** **T e** **s t** **o s** **t e** **r o** **n e** **T e** **s t** **e s** **W e** **i g** **h t** **o r** **M a** **l e** **F i** **s h** **G S** **I G** **o n** **a d** **S t** **a g** **i n** **g a** **n d** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **E p** **i d** **i d** **y m** **i d** **e s** **W e** **i g** **h t** **E p** **i d** **i d** **y m** **i d** **e s** **H i** **s t** **o p** **a t** **h o** **l o** **g y** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t A** **c c** **e s** **s o** **r y** **S e** **x O** **r g** **a n** **W e** **i g** **h t** **s /** **2 °** **S e** **x C** **h a** **r a** **c t** **e r** **i s** **t i** **c s** **A g** **e a** **n d** **W e** **i g** **h t** **a t** **P P** **S F** **e c** **u n** **d i** **t y** **/ f** **e r** **t i** **l i** **t y** **V i** **t e** **l l** **o g** **e n** **i n** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d 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 thy roid regulation. The various targets of the th yroid pathway are delineated so as to facilitate determination of potential for thy roid or HPT axis effects.
The EDSP Tier 1 battery

includes male and female pubertal rat studies with thy r oid endpoints and an amphibian metamorphosis assay (OCSPP 890.1100) for screening chemicals with the potential to interact with the thy roid pathway

. While Chemical X does not have an y Tier 1 screening submissions , several studies discussed in the case rev iew included endpoints that may provide anecdotal information about the potential to interact with the th yroi d pathway

. No equivalent EDSP Tier 1 assay s designed to evaluate potential interaction with T were available for this case stud y. Nagao et al. (2 000) reported increased thy roid and pituitary

weights and altered plasma T 3 , T 4 and TSH. When treated over two generations, TSH was significantl y increased, along with thy roid gland weight, in adults F 0 males exposed to the highest dose ( 50 mg/kg/d) of Che mical X, and significantl y decrease d in F 1 PND 22 males and females exposed to low (2 mg/kg/d) and high doses. T 3 and T 4 were significantl y decreased in F 0 adults treated with low concentrations of Chemical X, and concentrations remained low in males expo sed to high dose, though T 3 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. I t is worth noting that Chemical X display s clear and somewhat consistentl y non - linear dose

- response effects in the E and A pathway s, so the th yroid effects summarized in this case stud y ma y be a result of exposure to the chemical.
Interactions with the T pathway are not clearl y delineated b y the data summarized in this case study , however the studies reviewed not include assay s specifically d esigned to detect potential chemical interaction with the thy roid. Because of the limited data and ambiguous results, the potential of Chemical X to interact with the th yroid hormone pathway cannot be excluded.**Table 8** 

. Thy roid Pathway
**Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway**  **a**

**Study Type / Literature Citation** **H o** **r m** **o n** **e s** **( T** **4 a** **n d** **T S** **H )** **P i** **t u** **i t** **a r** **y W** **e i** **g h** **t T** **h y** **r o** **i d** **W e** **i g** **h t** **T h** **y r** **o i** **d G** **r o** **s s** **a n** **d H** **i s** **t o** **p a** **t h** **o l** **o g** **y F** **r o** **g D** **e v** **e l** **o p** **m e** **n t** **S t** **a g** **e H** **i n** **d L** **i m** **b L** **e n** **g t** **h S** **n o** **u t** **t o** **V e** **n t** **L e** **n g** **t h** **O v** **e r** **t T** **o x** **i c** **i t** **y O** **b s** **e r** **v e** **d b** 

**EDSP Tier 1** Male Rat ↑ (F o adult) ↓ (F 1 juv) ↑ ↑ M altered Female Rat ↑ (F 1 juv) T 3 - - -

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 o f overt toxicity c While overt toxicity was observed in this assa y 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 thyr oid 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 alway s reported. The degree of side chain branching may influence the affinit y of Chemical X for the ER , and perhaps other potential endocrine interactions. Chemical X appears to inhibit aromatase enzy me activit y and may alter transcripts of steriodogenic enz ym es upstream to estrogen s yn thesis. In vitro AR binding assay s and androgen receptor transactivation assay s 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 vi vo assay s 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 inter action in the thy roid endocrine pathway , though limited data suggest potential interaction with the mammalian thy roid s ys tem . Though results discussed in this evaluation must be regarded carefully and do not indicate clear relationships , the potential of Chemical X with the thy

roid hormone pathway cannot be discounted

.

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