No
                                     Effect

 Female Pubertal Rat

                                    ↓ 24%
                                     ↓43%
                                   No effect
                                    ↓ 35%
                                   No effect
                        ↑ 2-7 days (V0); ↓ 7-12% BW

                                      X[d]

 FSTRA

                               ↓ (13%) ♂ H[e]
                               ↑(90%) ♂ H[e]
                               ↑(39%) ♂ H[e]
                                       X
                                     OSRI
 Subchronic toxicity (Rat)

 Chronic toxicity (Rat)

                                   No effect
                                   No effect
                                   No effect
                                   No effect

 Chronic toxicity (Mouse)

                                   No effect

 Two-generation reproduction  (Rat)

                                   No effect
                                   No effect
                                   No effect
                                   No effect

                                   No effect

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 is not routinely evaluated or is not applicable in the assay.
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.
d Effects at the high dose (75 mg/kg/day) occurred in the presence of overt toxicity.  Effects at the low dose (10 mg/kg/day) were in the absence of overt toxicity.
e A 50% decrease in survival was observed at this treatment level.

Effects on Androgen Pathway: Table 41 below summarizes the results of the available EDSP Tier 1 screening assay results and OSRI results relevant for determining the potential for Chemical S to interact with the androgen pathway.  The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic, or HPG axis effects.
Chemical S was classified as a non-binder in the in vitro Androgen Receptor Binding assay (rat prostate cytosol), negative in the in vitro Steroidogenesis assay (human cell line H295R) and equivocal in the in vitro Aromatase assay (human recombinant microsomes).  Thus, the in vitro assays do not provide evidence of a specific androgen mode of action.  There is evidence that Chemical S has the potential to interact with the androgen pathway as shown in the in vivo Hershberger and the Male Pubertal assays.
In the anti-androgenic phase of the Hershberger assay, significant decreases in two (ventral prostate and glans penis) of the five weights examined were observed in male rats treated with 10 mg/kg/day in the absence of overt toxicity.  In addition, doses of 40 and 75 mg/kg/day Chemical S induced decreases in seminal vesicles, ventral prostate, LABC, Cowper's gland and glans penis weights in the presence of overt toxicity that was manifested as salivation (4 out of 6 rats at 40 mg/kg bw/day and 6 out of 6 rat at 75 mg/kg bw/day) and decreased terminal body weights and body weight gains at 75 mg/kg bw/day.  In the androgenic phase of the assay, while no treatment-related effects were seen in androgen sensitive tissues, clinical signs of toxicity (salivation) were seen in 3 of 6 rats.
In the Male Pubertal assay, responses relevant to interaction with the androgen pathway consistent with antagonism included delayed preputial separation, decreases in the weights of seminal vesicles, ventral prostate, dorsolateral prostate, LABC, epididymides, adrenals, and testes; and decreased testosterone levels, but these responses were seen only at a dose (75 mg/kg bw/day) that was associated with overt toxicity (salivation in all treated rats; significant decreases in body weights and food consumption).  As discussed in the E pathway discussion above, given, the decreases observed in the endpoints for the male pubertal are consistent with Chemical S's mode of action (i.e., depletion of ATP and caloric intake). In the FSTRA assay, potential endocrine effects (e.g., alterations in fertility, males GSI, and male tubercles) were observed only at levels with overt toxicity (increased mortality). However, as discussed above, there is uncertainty in whether these responses are due to a potential endocrine-interaction or from a response in decreased energy output.
Increased testes weights (absolute and relative) were noted in the Part 158 chronic toxicity study in the rat; however, there were no accompanying histological effects.  In the Male Pubertal assay, decreased rather than increased testes weights were observed, but the decreased testes weights occurred at a dose level associated with overt toxicity. Again, effects on reproduction were reported in the bird and invertebrate studies, but the changes reported lack endocrine-specificity.
Based upon WoE evaluation of EDSP Tier 1 screening assay and OSRI results, in the absence of overt toxicity, Chemical S demonstrated a potential to interact with the androgen pathway (androgen antagonism) in only the Hershberger assay however, this impact is not supported by information provided through the other relevant assays.

Table 41.  Androgenic/Anti-Androgenic Pathway for Chemical S
 Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chemical S[a]
                        Study Type/Literature Citation

 AR Binding
 Steroidogenesis
 Testosterone
 Testes Weight
 Gonad Staging and Histopathology
 Epididymides Weight
 Epididymides Histopathology
 Pituitary Weight
 Accessory Sex Organ Weights/2° Sex Characteristics
 Age and Weight at PPS
 Vitellogenin
 Fish Gonadal Weight (GSI)
 Overt Toxicity Observed[b]
                                  EDSP Tier 1
AR Binding
                                  Non-binder

Steroidogenesis

                                   Negative

Hershberger

                                   ↓16-40%

                                     X[c]
Male Pubertal Rat

                                    ↓56%
                                    ↓17%
                                   No effect
                                    ↓24%
                                   No effect
                                   No effect
                                   ↓25-55%
                           ↓11% BW; ↑ 6 d (PPS)

                                     X[d]
FSTRA

                               ↓(33%) ♂ H[e]

                               ↑(39%) ♂ H[e]

                                       X
                                     OSRI
Subchronic toxicity (Rat)

                                   No effect
                                   No effect

Chronic toxicity (Rat)

                                    ↑27%
                                   No effect

                                   No effect

Chronic toxicity (Mouse)

                                   No effect
                                   No effect

Chronic toxicity
(Dog)

                                   No effect

Two-generation reproduction  (Rat)

                                   No effect
                                   No effect

                                   No effect
                                   No effect
                                   No effect

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 is not routinely evaluated or is not applicable in the assay.
b An"X" in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity.
c Effects at the mid  -  and high dose (40 and 75 mg/kg/day) occurred in the presence of overt toxicity.  Effects (ventral prostate and glans penis weights) at the low dose (10 mg/kg/day) were in the absence of overt toxicity in the anti-androgen phase of the assay.
d Effects at the high dose (75 mg/kg/day) occurred in the presence of overt toxicity.  There were no effects at the low dose (10 mg/kg/day).
e A 50% decrease in survival was observed at this treatment level.

     i. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis
In general, regulation of the HPT axis is comparable to the HPG axis, except that the feedback relationship involves thyroid hormones (e.g., T4 and T3).  Thyroid hormones feedback to the hypothalamus or pituitary to regulate thyrotropin-releasing hormone and TSH, respectively, which, in turn, regulate hormone production by the thyroid gland.  The current Tier 1 screening battery does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it does include three in vivo assays that have the potential to detect these effects on the HPT axis (i.e., the pubertals and the AMA).
Thyroid Hormones: Table 42 below summarizes the results of the available EDSP Tier 1 screening assay and OSRI results for determining the potential for Chemical S to interact with thyroid regulation. The various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects.

Table 42.  Thyroid Hormone Pathway for Chemical S
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical S[a]

Study Type/Literature Citation
Hormones         (T4 and TSH)
Pituitary Weight
Thyroid Weight
Thyroid Histopathology
Frog Development Stage
Hind Limb Length
Snout to Vent Length
Overt Toxicity Observed[b]
EDSP Tier 1
Male Pubertal (Rat)
No effect
No effect
No effect
No effect

X
Female Pubertal (Rat)
No effect
↓ 35%
↓ 14%
No effect

X
AMA (Frog)[c]

OSRI
Two-generation reproduction  (Rat)

No effect

Subchronic toxicity (Rat)

Chronic toxicity (Rat)

No effect
No effect
No effect

Chronic toxicity (Mouse)

No effect
No effect
No effect

Chronic toxicity (Dog

No effect

  a   Key to responses:  Positive (P), Negative (N) or Equivocal (E) observation; arrows (↓ or ↑) indicate the direction of the response; -- indicates that parameter was not evaluated or is not applicable.
  b   An "X" in this column indicates that the effect(s) observed in the assay occurred in the presence of overt toxicity.
  c   Exposure may not have been inadequate to elicit a response.

There was no evidence that Chemical S has a potential to interact with the thyroid hormonal pathway.   The decreased thyroid and pituitary weights observed in the Female Pubertal assay at 75 mg/kg bw/day occurred in the presence of overt toxicity (salivation and decreases in body weights and food consumption).  There were no effects on the thyroid in the standard combined chronic toxicity/carcinogenicity studies in the rat and mouse.
Adequate exposure was not achieved in the Amphibian Metamorphosis assay (AMA), and therefore was not successfully tested in this assay.  Testing in the AMA assay is limited by the physico-chemical characteristics of the test substance.  With respect to Chemical S, it belongs to a class of chemicals characterized by relatively low water solubility, high octanol-water partition coefficients (Kow) and high organic carbon partition coefficients (Koc).  While these compounds are relatively stable to both abiotic (hydrolysis and photolysis) and biotic (aerobic and anaerobic) degradation/metabolism, their low solubility and propensity to partition to organic matter render them difficult to test in aquatic systems.
Based upon WoE evaluation of EDSP Tier 1 screening assay and OSRI results, Chemical S did not demonstrate a potential to interact with the thyroid hormone pathway.

Conclusions for Chemical S
All eleven assays comprising the EDSP Tier 1 battery were available for Chemical S. Chemical S was evaluated for several hypothesized endocrine modes of action for each specific E, A, and T pathway including agonism and antagonism at the estrogen and androgen receptor, altered steroidogenesis, HPG and HPT perturbations.  In addition to the available Tier 1 data, other relevant studies of sufficient quality were considered in this assessment. These studies included standard Part 158 toxicity data relevant to evaluating effects in endocrine tissues and functions.
In determining whether Chemical S interacts with E, A, or T hormonal pathways,  the  number and type of effects induced, the magnitude of responses, and the  pattern of responses observed across studies, taxa, and sexes were considered.  Additionally, the conditions under which effects occur were considered, in particular, the dose(s) at which responses happen and whether overt toxicity was present. This is particularly important in the case of Chemical S, given that it is a severe irritant to the gastrointestinal tract (see section <IP_ADDRESS>. Discussion of the Toxicity Profile in the Context of Tier I Assays).  Thus, studies at doses levels at which potential irritation would occur were considered to evaluate sources of potential overt toxicity.   Additionally, as the chemical's mode of action eventually results in a depletion of ATP, consideration was given in how this may have impacted the responses observed in the studies.
Integrating the in vitro and in vivo Tier 1 results, as well as consideration of relevant data beyond Tier 1 information (e.g., Part 158 standard toxicology studies) revealed evidence of a potential interaction with the E or A pathways.  In the absence of overt toxicity, there was evidence of potential interaction with the androgen signaling pathway.  The complementary responses in the Hershberger assay were limited to significant decreases in only two androgen-dependent tissues.  Although androgen-related responses were seen in the male pubertal assay and FSTRA, they occurred in the presence of overt toxicity, and the directionality of the reported effects was consistent with Chemical S's mode of action.  Thus, there were no redundant responses across the in vivo assays to provide confirmatory evidence.  Likewise, Chemical S demonstrated a potential to interact with the estrogen signaling pathway in that only one response (delayed vaginal opening in the female pubertal assay) was significant.  Although there were responses in FSTRA that could be indicative of an interaction with the E signaling pathway, these responses were only observed in the presence of overt toxicity, which again could be due to the lack of energy reserves for input into reproductive status.  The negative complementary and redundant responses in the rat pubertal assay and AMA indicates that Chemical S does not interact with the thyroid hormonal pathway.

        Case Study  --  Chemical J

B. Introduction
Chemical J is a triazole fungicide inhibiting sterol biosynthesis which in turn disrupts membrane function.  Chemical J also acts as a hepatic microsomal enzyme inducer.  In the case of Chemical J, issues included: Tier 1 responses that may be reflective of potential interactions with perturbation of more than one endocrine modality (i.e., EAT) and different effects across taxa which may have implications for Tier II testing.
The available information considered to determine the potential interaction of Chemical J with the E, A and/or T pathways include all 11 EDSP Tier 1 studies and other scientifically relevant information (OSRI) such as 40CFR Part 158 guideline toxicity studies.  The data considered are summarized in Sections B, C and D of this chapter.  An analysis of the data submitted to the agency, using the WoE approach outlined by the agency (USEPA, 2011b), is presented in Section E.

Data Available for Chemical J
Chemical J is soluble in water and does not volatize appreciably. It 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 compound can slowly undergo aerobic metabolism with half-lives >70 days.  Based on a log octanol-water partition coefficient (log Kow) of approximately 3 and a relatively low propensity to sorb to organic matter, based on its organic carbon partition coefficient (Koc~520 mL/goc), Chemical J residues are not expected to bioaccumulate.

Tier 1 Screening Assays for Chemical J
The Tier 1 studies submitted in response the test order for Chemical J are shown below in Table 43 and findings are summarized below.
Table 43. Summary of Tier 1 Screening Assays for Chemical J
Tier 1 Assays Studies
Test Guideline
Satisfied by OSRI (Y/N)

Study Findings
ER Binding Assay (Rat uterine cytosol)

OSCPP 890.1250
No
Negative[1]
ERα Transcriptional Activation Assay (Human cell line HeLa 9903)

OSCPP 890.1300; OECD 455
No
Negative
AR Binding Assay (Rat prostate cytosol)

OSCPP 890.1150
No
Positive[2]
Steroidogenesis Assay (Human cell line H295R)

OSCPP 890.1550; OECD 456
No
Positive
Aromatase Assay (human recombinant microsomes)

OSCPP 890.1200
No
Positive[3]
Uterotrophic Assay (Rat)
OSCPP 890.1600; OECD 440
No
Negative
Hershberger Assay (Rat)
OSCPP 890.1400; OECD 441
No
Negative for Androgenic and Anti-androgenic
Pubertal Female Assay (Rat)

OSCPP 890.1450
No
Negative for E and T pathways
Pubertal Male Assay (Rat)

OSCPP 890.1500
No
Positive for A pathway
Negative for T pathway
Fish Short-term Reproduction Assay

OSCPP 890.1350; OECD 229
No
Positive for E and A pathway
Amphibian Metamorphosis Assay (Frog)
OSCPP 890.1100; OECD 231
No
Negative
      1 Classified as "Not Interactive" according to the Test Guideline OSCPP 890.1250
      2 Classified as "Binder" according to the Test Guideline OSCPP 890.1150
3Classified as "Inhibitor" according to the Test Guideline OSCPP 890.1200

              i.               ER Binding Assay (Rat uterine cytosol)
In the ER binding assay, uterine cytosol from Sprague Dawley rats was used as the source of ER to conduct the binding assays. A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the ER in the isolated uterine cytosol was present in an adequate concentration and functioning with the appropriate affinity for the radiolabeled (tritium; [3]H) ligand.  The competitive binding experiment was conducted to measure the binding of a single concentration of [[3]H]-17β-estradiol (1 nM) in the presence of increasing concentrations of Chemical J (logarithmic increase from 10[−10] to 10[−3] M).  Ethanol was used as the vehicle at a final concentration of <3% (v/v).  The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference material.
In the saturation binding experiment, the maximum binding capacity (Bmax) was 59.28 fmol/100 ug protein and the dissociation constant (Kd) was 0.1032 nM.  The performance criteria were met for the saturation binding assay.
In the competitive binding experiment, Chemical J did not competitively bind to the ER at concentrations up to 10[−3] M.  Across all three runs, specific binding averaged 94.5  -  102.9% at Chemical J concentration of 10[−10] to 10[−3] M.  The mean specific binding affinity (IC50) and relative binding affinity (RBA) were not calculated for Chemical J because it did not achieve >=50% displacement of [[3]H]-17β-estradiol.
The performance criteria were met for the competitive binding experiment and the reference compounds generally performed as expected.  The negative control, octyltriethoxysilane, had no effect on binding, and there were concentration-dependent decreases in binding for the natural ligand and the weak positive control.
Based on the results of three runs, Chemical J is classified as Not Interactive in the ER binding assay.

              ii. ER Transcriptional Activation Assays
In the ER transcriptional activation assay, hERα-HeLa-9903 cells cultured in vitro were exposed to Chemical J at logarithmically increasing concentrations from 10[−10] to 10[−4] M in dimethylsulfoxide (DMSO; 0.1%) for 24 hours in three independent runs.  The experiments were performed using 96-well plates and each Chemical J concentration was tested in triplicate (3 wells/plate) in each run.  Cells were exposed to the test agent for approximately 24 hours to induce reporter (luciferase) gene products.  Luciferase expression in response to activation of the estrogen receptor by Chemical J was measured upon addition of a luciferase substrate and detection with a luminometer.
Chemical J was tested up to the limit of solubility, 10[−4] M.  The maximum level of response (RPCmax) was 6.4% for the first run, 2.8% for the second run and 7.3% for the third run, with the associated maximum level of response (PCmax) of 10[−5] M for all three runs.
In runs 1 and 3, all performance criteria were met for the reference chemicals 17 β-estradiol, 17α-estradiol, and 17 α-methyltestosterone.  The Hill Slope values for 17 β-estradiol (0.4) and  17α-estradiol (0.6) in run #2 fell outside the expected ranges (0.7 to 1.5 for 17 β-estradiol; 0.9 to 2.0 for 17α-estradiol).  The Log PC50 and Log PC10 values for 17 α-methyltestosterone were lower than the expected values for all three runs.  The Log PC50 values for each run were -7.6, -6.6 and -6.1 (expected range -6.0 to -5.1) and the LogPC10 values for the runs were -8.8, -8.9 and -8.8 (expected range -8.0 to -6.2).  These minor deviations did not negatively affect the interpretation of the study.
Chemical J was negative for ER transcriptional activation in this test system.

      iii.  AR Binding Assay (Rat prostate cytosol)
In the AR binding assay, ventral prostate cytosol homogenate from Sprague Dawley rats was used as the source of AR to conduct the binding assays.  A saturation binding experiment was conducted prior to the competitive binding experiment to demonstrate that the AR in the cytosol preparation was present in an adequate concentration and functioning with the appropriate affinity for the radiolabeled ([3]H) ligand.  The competitive binding experiment, was conducted to measure the binding of a single concentration of the androgen ligand [[3]H]-R1881 (1 nM) in the presence of increasing concentrations of Chemical J (logarithmic increase from 10[−10] to 10[−3] M).  Ethanol was used as a vehicle at a final assay concentration of <3% (v/v).  The assay included dexamethasone as a weak positive control, and unlabeled R1881 as the ligand reference standard.
In the saturation binding experiment in which two runs were conducted, the maximum binding capacity (Bmax) was 3.245 fmol/100 ug protein and the dissociation constant (Kd) was 0.4641nM.  Although the Bmax fell slightly below the expected range of 10 to 150 fmol/100 ug protein and the Kd was also below the expected range of 0.685 to 1.57 nM, confidence in these results is high because of the goodness-of-fit (r[2] = 0.9871  -  0.9937) and the reproducibility between the runs.  With the exception of the high concentration of 10 nM in one assay with a 24.6% nonspecific binding, non-specific binding as a percent of total binding was <20% across the entire concentration range tested for the saturation binding assay.  All other performance criteria and the competitive binding assays indicated acceptable performance of the assay.
In the competitive binding experiment, Chemical J reduced the mean specific binding of the radiolabeled ligand at the highest two concentrations in a concentration-dependent manner; however, binding curve decreased from approximately 84% to approximately 14% over a single log unit of concentration (from 10[-4] to 10[-3] M) and had steep Hill slopes in runs 2 and 3.  The initial analysis suggested that the data for runs 2 and 3 did not fit the four-parameter, nonlinear regression model for analysis of one-site binding.  However, these results may be an artifact of the data when using an undefined bottom plateau for the curve fitting.  When the data were re-analyzed by constraining the bottom of the response curves to zero, the results of all three independent runs are similar with acceptable r[2] values and standard errors.
The mean estimated log IC50 was -3.5 M for the analysis using a constrained bottom plateau.  The range of RBA for values for Chemical J across the three runs was 0.00034% to 0.00037% indicating a weak interaction with the AR.  The mean Log IC50 values for the strong positive control R1881 and the weak positive control (dexamethasone) were -9.0 and -4.4 M, respectively, and the mean RBA for the weak positive control was 0.0028%.  All performance criteria for the competitive binding assays indicated acceptable performance of the assay.
The results from the 3 competitive binding experiments, with the undefined bottom plateau, are summarized in
Table 44 and shown graphically in Figure 14.  The results when constraining the plateau are presented in Table 45 and Figure 1.
Based on the results of three runs, Chemical J is classified as a "Binder", albeit with a weak affinity, in the AR binding assay.

Table 44:  Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (using undefined bottom plateau)
                                   Parameter
                                     Run 1
                                     Run 2
                                     Run 3
                                     Mean
                                            r[2] (unweighted)             R1881
                                    0.9999
                                    1.0000
                                    0.9999
                                      NA
                                                               Positive control
                                    0.9999
                                    0.9997
                                    0.9995
                                      NA
                                                                     Chemical J
                                    0.9922
                                    0.9969
                                    0.9988
                                      NA
Log IC50 (M)           R1881
                                    -8.986
                                    -8.958
                                    -8.994
                                    -8.978
                                                               Positive control
                                    -4.440
                                    -4.382
                                    -4.455
                                    -4.426
                                                                     Chemical J
                                    -3.419
                                   ~-3.830c
                                   ~-3.855c
                                    ~-3.848
IC50 (M)                   R1881
                                 1.032 x 10-9
                                 1.101 x 10-9
                                 1.014 x 10-9
                                 1.052 x 10-9
                                                               Positive control
                                 3.630 x 10-5
                                 4.154 x 10-5
                                 3.504 x 10-5
                                 3.750 x 10-5
                                                                     Chemical J
                                3.810 x 10[-4]
                               ~1.480 x 10[-4b]
                              ~1.395 x 10[-4(b)]
                                ~1.420 x 10[-4]
Log RBA[(a)] Positive control
                                     -2.6
                                     -2.6
                                     -2.5
                                     -2.6
                                                                     Chemical J
                                     -3.6
                                      NA
                                      NA
                                      NA
                                                       RBA (%) Positive control
                                    0.0028
                                    0.0027
                                    0.0029
                                    0.0028
                                                                     Chemical J
                                    0.00027
                                      NA
                                      NA
                                      NA
a Calculated by the reviewer.
b Approximate estimates as 95% CI estimates were very wide
NA = Not applicable;
r[2] = Goodness of fit;
RBA (%) = relative binding affinity

Figure 14.  Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Chemical J (Assays 1  -  3), Using an Undefined Bottom Plateau.
                                   Assay #1

                                   Assay #2

                                   Assay #3

Table 45: Competitive Binding Assay of Chemical J with AR from Rat Prostate Cytosol (using confined bottom plateau)

                                   Parameter
                                     Run 1
                                     Run 2
                                     Run 3
                                     Mean
r[2] (unweighted)	    Chemical J
                                    0.9819
                                    0.9902
                                    0.9939
                                      NA
Log IC50 (M)	           Chemical J
                                    -3.552
                                    -3.486
                                    -3.493
                                    -3.510
IC50 (M)	          Chemical J
                                2.805 x 10[-4]
                                3.266 x 10[-4]
                                3.214 x 10[-4]
                                3.112 x 10[-4]
RBA (%)	           Chemical J
                                    0.00037
                                    0.00034
                                    0.00033
                                    0.00035
a Calculated by the reviewer.
NA = Not applicable; r[2] = Goodness of fit; RBA (%) = relative binding affinity

Figure 15.  Percentage R1881 Bound to the Androgen Receptor in the Presence of Radioinert R1881, Dexamethasone, and Chemical J (Assays 1  -  3), Using a Constrained Bottom Plateau, for Competitive Runs 1, 2, and 3.

              iii. Steroidogenesis Assay (Human cell line H295R)
In the steroidogenesis assay, H295R cells cultured in vitro in 24-well plates were incubated with Chemical J at log concentrations from 10[−10] to 10[−4] M for 48 hours in triplicate in three independent experiments.  The test chemical's vehicle was DMSO, at a final concentration of 0.1% (v/v).  Testosterone and estradiol levels were measured using liquid chromatography coupled with atmospheric pressure photoionization tandem mass spectroscopy (LC-APPI-MS/MS).  A Quality Control (QC) plate was run concurrently with each independent run of a test chemical plate to demonstrate that the assay responded properly to positive control agents at two concentration levels.  The positive controls included a known inhibitor (prochloraz) and a known inducer (forskolin) of estradiol and testosterone production.
Chemical J was not cytotoxic to the H295R cells up to the test limit concentration.  Viability in each run of the test plate ranged from 98 - 129% relative to solvent controls.  Viability in each run of the QC plate was 84 - 127% relative to solvent controls, except for the methanol-treated wells which were 27 - 32%.
Testosterone levels were decreased (p<=0.05) to 0.3 - 0.4-fold of the solvent control values in all 3 runs at 100 uM for Chemical J.  In Run 2, there were significant increases in testosterone production (1.3- to 1.5-fold change) at concentrations from 0.01 to 10 uM; however, the induction was not reproducible in the other two runs.
Decreased (0.8-fold the solvent control; p<=0.05) estradiol levels were observed at 0.1 uM for Chemical J in runs 2 and 3, and significant decreases were observed in all 3 runs at concentrations >=1 uM (0.4 - 0.7-fold; p<=0.05).  Decreased (p<=0.05) estradiol (0.8-fold the solvent control) was also noted at the lowest level tested (0.0001 uM) in Run 2.  The effects of Chemical J on testosterone and estradiol production are summarized in Table and shown in Figure 16 and Figure 17.
Guideline acceptability recommendations and requirements were generally met, including lack of cytotoxicity, adequate production of testosterone and estradiol, acceptable reproducibility (i.e., low percent coefficients of variability (%CV), and appropriate induction and inhibition with positive controls.  The exceptions included:  (i) the testosterone level in the solvent control (SC) of run 2 was 470 pg/mL (< the 500 pg/mL guideline limit); (ii) the estradiol level in the SC of run 1 was 35.5 pg/mL (< the 40 pg/mL guideline limit); and (iii) the inhibition of estradiol in run 1 with 1 uM prochloraz was 0.6-fold (> the 0.5-fold guideline limit). However, these exceptions were slight and did not impact the interpretation of the results.
Based on the hormone responses in three runs, Chemical J was positive in the steroidogenesis assay.

Table 46:  Mean (+-SD) Hormone Concentrations following Treatment with Chemical J for 48 Hours.[a]
                          Nominal Concentration (uM)
                                     Run 1
                                     Run 2
                                     Run 3
                                     Run 1
                                     Run 2
                                     Run 3
                                     Mean
                                     +- SD
                           Statistical Significance

                             Testosterone (pg/mL)
                                Fold Difference
                                     DMSO
                                     616.3
                                     461.0
                                     631.3
                                       --
                                       --
                                       --
                                       --
                                       --
                                       --
                                    0.0001
                                     512.3
                                     497.3
                                     540.0
                                      0.8
                                      1.1
                                      0.9
                                      0.9
                                      0.1
                                       --
                                     0.001
                                     547.3
                                     592.3
                                     576.7
                                      0.9
                                      1.3
                                      0.9
                                      1.0
                                      0.2
                                    Run #2
                                     0.01
                                     564.3
                                     621.0
                                     572.7
                                      0.9
                                      1.3
                                      0.9
                                      1.1
                                      0.3
                                    Run #2
                                      0.1
                                     613.0
                                     640.0
                                     603.3
                                      1.0
                                      1.4
                                      1.0
                                      1.1
                                      0.2
                                    Run #2
                                       1
                                     608.7
                                     674.0
                                     690.3
                                      1.0
                                      1.5
                                      1.1
                                      1.2
                                      0.2
                                    Run #2
                                      10
                                     679.7
                                     647.0
                                     726.7
                                      1.1
                                      1.4
                                      1.2
                                      1.2
                                      0.2
                                       --
                                      100
                                     196.7
                                     204.7
                                     199.0
                                      0.3
                                      0.4
                                      0.3
                                      0.4
                                      0.1
                                  All 3 Runs

                               Estradiol (pg/mL)
                                Fold Difference
                                     DMSO
                                     42.0
                                     49.2
                                     57.3
                                       --
                                       --
                                       --
                                       --
                                       --
                                       --
                                    0.0001
                                     38.1
                                     41.0
                                     55.9
                                      0.9
                                      0.8
                                      1.0
                                      0.9
                                      0.1
                                    Run #2
                                     0.001
                                     39.0
                                     46.3
                                     55.2
                                      0.9
                                      0.9
                                      1.0
                                      0.9
                                      0.0
                                       --
                                     0.01
                                     37.6
                                     45.5
                                     53.8
                                      0.9
                                      0.9
                                      0.9
                                      0.9
                                      0.0
                                       --
                                      0.1
                                     34.8
                                     39.5
                                     45.2
                                      0.8
                                      0.8
                                      0.8
                                      0.8
                                      0.0
                                 Run #2 and #3
                                       1
                                     22.8
                                     35.0
                                     29.2
                                      0.5
                                      0.7
                                      0.5
                                      0.6
                                      0.1
                                  All 3 Runs
                                      10
                                     19.1
                                     17.6
                                     21.3
                                      0.5
                                      0.4
                                      0.4
                                      0.4
                                      0.1
                                  All 3 Runs
                                      100
                                     28.9
                                     28.9
                                     33.3
                                      0.7
                                      0.6
                                      0.6
                                      0.6
                                      0.1
                                  All 3 Runs
a The lower limit of quantification (LLQ) was 25 pg/mL for testosterone and 10 pg/mL for estradiol.

Figure 16.  Change in Testosterone Production Relative to Chemical J Concentration.

Figure 17.  Change in Estradiol Production Relative to Chemical J Concentration.

              iv. Aromatase Assay (Human Recombinant Microsomes)
In the in vitro aromatase (CYP 19) assay, Chemical J was incubated with human recombinant aromatase and tritiated androstenedione (1-β [[3]H(N)]-androst-4-ene-3,17-dione ([[3]H]ASDN)) in ethanol for 15 minutes to assess the potential of Chemical J to inhibit aromatase activity.  Chemical J was tested at logarithmic concentrations from 10[ - 10] M to 10[−3] M in 3 independent runs.
Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a 15-minute incubation for each concentration of chemical.  Tritiated water was quantified using liquid scintillation counting.  Three runs were conducted and each run included a full activity control, a background activity control, a positive control series (10[ - 10] to 10[ - 5] M)) with a known inhibitor (4-OH ASDN), and the test chemical series (10[ - 10] to 10[ - 3] M) with 3 repetitions per concentration.  Aromatase activity in the full activity controls ranged from 0.090 to 0.174 nmol∙mg-protein[−1]∙min[−1] for the three test runs, with a mean and standard deviation of 0.136+-0.016 nmol∙mg-protein[−1]∙min[−1].  Activity in the background controls ranged from 0.13% to 0.60% of the overall average of the background-adjusted full activity controls.  The responses of the full activity and background controls were acceptable.
For the positive control substance (4-OH ASDN), aromatase activity averaged 0.145+-0.017 nmol∙mg-protein[−1]∙min[−1] at the lowest tested concentration (10[−10 ]M) and 0.0016+-0.0001 nmol∙mg-protein[−1]∙min[−1] at the highest tested concentration (10−5 M).  These results were within the recommended ranges for the top of the curve, bottom of the curve, Hill slope, Log IC50, and CV for replicates of each concentration, with the exception of some minor deviations for Run #1.  The minor deviations in Run 1 included a % top of curve of 111% (criterion:  90-110%) and a LogIC50 of -6.9 (criterion -7.3 to 7.0).  All performance criteria were met in Run #2 and #3, with the mean performance criteria values within the recommended ranges.
For Chemical J, aromatase activity averaged 0.127+-0.013 nmol∙mg-protein[−1]∙min[−1] at the lowest tested concentration (10[−10 ]M) and 0.00017+-0.00004 nmol∙mg-protein[−1]∙min[−1] at the highest tested concentration (10−3 M).  The data for Chemical J were modeled for all runs; the best fit Hill slope and Log IC50 values across the three independent runs did not differ significantly.  The mean goodness-of-fit (r[2]) value was 0.99, and the mean log IC50 of the test material was  - 6.6.  The average dose-response curve indicated that the aromatase activity of the test material at concentrations ranging from 10[−10] M to 10[−8] M was essentially equivalent to the activity observed in the full activity controls.  The 10[ - 6] M dose level reduced the activity by approximately 75%, and the 10[ - 4] and 10[ - 3] M dose levels reduced the activity by >99%.
The mean aromatase activity data for Chemical J (expressed as % full control activity) for each concentration tested across all 3 runs are presented in Table 47, along with the overall standard deviation and % CV.  Inhibition response curves for Chemical J from each run are shown in Figure 18.
Based on the results of three, Chemical J is classified as inhibitor in the aromatase assay.
Table 47:  Effect of Chemical J on Aromatase Activity (as Percent of Control) From Independent Runs [a]
Chemical
                                     Run 1
                                     Run 2
                                     Run 3
                                     Mean
                                      SEM
                                      %CV
                                 Log IC50 (M)
Chemical J
                                    −6.6
                                    −6.8
                                    −6.5
                                    −6.6
                                     0.09
                                    −2.30
4-OH ASDN
                                    −6.9
                                    −7.1
                                    −7.1
                                    −7.0
                                     0.07
                                    −1.64
                                  Hill Slope
Chemical J
                                    −0.92
                                    −0.71
                                    −0.87
                                    −0.83
                                     0.06
                                   −13.16
4-OH ASDN
                                    −0.92
                                    −1.0
                                    −0.96
                                    −0.96
                                     0.02
                                    −4.17
a = mean, SEM, and % CV estimates were calculated by the reviewer. SEM = Standard Error of the Mean.

Figure 18.  Aromatase Inhibition Response Curves for Chemical J from Each Test Run.

      vi. Uterotrophic Assay (Rat)
In the in vivo uterotrophic assay conducted to screen for potential estrogenic activity, Chemical J was administered daily in corn oil via oral gavage to groups of six immature female Sprague Dawley rats at dose levels of 0 (vehicle), 50, 150, or 475 mg/kg bw/day on post-natal days (PND) 19-21.  A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 10 μg/kg bw/day by oral gavage.  Body weights were determined daily.  All animals were examined for vaginal opening (VO), and then euthanized and necropsied on PND 22 approximately 24 hours after the final dosing to determine wet and blotted uterine weights.
All animals survived until scheduled termination.  No clinical signs of toxicity were observed, and no significant increase in VO was noted in the Chemical J-treated females.  At 475 mg/kg bw/day, body weights were decreased (p<0.05) by 15% on Day 3, resulting in a 59% decrease (p<0.05) in overall (Days 1-4) body weight gains relative to the vehicle control.  Uterine weights at this dose were comparable to the vehicle controls.  Absolute wet and blotted uterus weights for the positive control (EE) group were increased (p<0.05) by 891% and 524%, respectively.
The 59% decrease in overall body weight gains in the high-dose group indicated that these animals were dosed at adequate levels.
No statistically significant increase in uterine weight was seen in this study. Hence, Chemical J was negative in the Uterotrophic assay.

              i. Hershberger Assay (Rat)
In the in vivo Hershberger assay testing for androgenic activity, Chemical J was administered daily in corn oil via oral gavage to groups of seven 55-day old castrated male Sprague Dawley rats/group at dose levels of 0 (vehicle), 25, 125, or 500 mg/kg bw/day for 10 consecutive days.  The study included an androgenic positive control group of seven castrated male rats dosed daily with testosterone propionate (TP) at 0.4 mg/kg bw/day via subcutaneous (s.c.) injection.
To screen for possible anti-androgenic activity, additional groups of seven 55-day old castrated male Sprague Dawley rats/group were dosed daily via oral gavage with Chemical J in corn oil at 25, 125, or 500 mg/kg bw/day for 10 consecutive days in conjunction with a daily s.c. injection of TP at 0.4 mg/kg bw/day.  The control group consisted of seven castrated male rats dosed daily with TP (0.4 mg/kg bw/day) by s.c. injection, and the positive control group consisted of seven castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg bw/day for 10 days in conjugation with a daily s.c. dose of TP (0.4 mg/kg bw/day).
Body weights, body weight gains, and food consumption were measured.  Animals were euthanized approximately 24 hours after the final dose administration.  The five androgen-dependent tissues (i.e., seminal vesicles, ventral prostate, levator ani-bulbocavernosus (LABC), Cowper's glands or glans penis) as well as adrenals, kidneys, and liver tissues were weighed and macroscopically examined.
No treatment-related effects were observed in either the agonist or antagonist assays on mortality, clinical signs of toxicity, body weights, body weight gains, food consumption, adrenals and kidneys weights, or gross pathology. The increased liver weights and microscopic liver findings at 500 mg/kg bw/day indicated that the animals were dosed to a level that showed toxicological effects, i.e., overt toxicity.
For the androgenic portion of the study, there were no statistically significant increases in the weights of accessory sex tissue at any dose of Chemical J.  For the TP positive control group, there were significant (p<0.05) increases in the weights of the Cowper's glands (688%), glans penis (77%), LABC (194%), ventral prostate (917%), and seminal vesicles with coagulating glands (1105%), indicating that the test system was sensitive to an androgenic response.
For the anti-androgenic portion of the study, there was a statistically significant decrease in one of the five target accessory sex tissues.  A significant 20% decrease (p<0.05) in LABC weight was observed at 500 mg/kg bw/day of Chemical J + TP.  No significant changes in organ weights were seen in the seminal vesicle, ventral prostate, Cowper's gland or glans penis as any dose. The decrease in LABC weight at 500 mg/kg bw/day is not considered a positive result for anti-androgenic activity in this assay as only a single organ weight was decreased.  For the TP+FT positive control group, there were treatment-related decreases in the weights of the Cowper's glands (62%), glans penis (26%), LABC (54%), ventral prostate (75%), and seminal vesicles with coagulating glands (81%) relative to weights in the "TP only" treated group.  These data indicate that the test system was sensitive to an anti-androgenic response.
Liver weights were increased by Chemical J treatment in both the androgenic and anti-androgenic portions of the study.  For the androgenic portion of the study, absolute liver weights were increased by 20% and 53% at 125 and 500 mg/kg bw/day Chemical J, respectively.  For the anti-androgenic portion of the study, there was a 59% increase in absolute liver weight in animals given 500 mg/kg bw/day with TP.  These weight increases correlated with histopathologic lesions in the liver, which included hepatocellular hypertrophy, vacuolization, and necrosis.
The performance criteria for the assay were met as the %CVs for weights of accessory sex tissues in the control and treated groups were below the maximum permissible values specified in the guideline.  No statistically significant changes were seen in two or more of the androgen responsive tissues.
Chemical J was negative for androgenicity and anti-androgenicity in the Hershberger assay.
              ii. Pubertal Female Assay (Rat)
In a Female Pubertal Assay, 16 Sprague Dawley [Crl:CD(SD)] rats/dose group were treated daily via oral gavage with Chemical J in corn oil at doses of 0, 50, 200, or 400 mg/kg bw/day from post-natal day (PND) 22 to 42.  Animals were examined for vaginal opening (VO) daily beginning on PND 22, and weight at day of attainment was recorded.  Following sacrifice on PND 42, total thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using chemiluminescent immunoassay and radioimmunoassay, respectively.  At termination, weights were measured for liver, kidneys, thyroid, adrenals, pituitary, and urogenital organs, and microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys from the control and high-dose groups and on livers from all dose groups.
At 400 mg/kg bw/day, treatment-related increases (p<0.05) were observed in alanine aminotransferase (ALT; ↑24%) and gamma glutamyltransferase (GGT; ↑130%).  Dose-dependent increases (p<0.05) in absolute, relative, and adjusted liver weights were observed at 200 (↑18-19%) and 400 (↑36-41%) mg/kg bw/day.  Animals in these dose groups also displayed treatment-related histopathological effects consisting of very slight to slight hepatocyte hypertrophy with altered tinctorial properties (increased cytoplasmic eosinophilia) of centrilobular and midzonal hepatocytes, very slight multifocal chronic inflammation, and very slight to slight increased number of mitotic figures of hepatocytes.  These findings showed a dose-dependent increase in incidence and severity, and were indicative of hepatotoxicity at 400 mg/kg bw/day.  Based on these findings in liver and on evidence of hepatic necrosis at 500 mg/kg bw/day in a concurrently submitted Hershberger study, the high dose in the current study appears to be an adequate high dose.
There were no significant treatment-related changes or alterations on mortality, clinical signs of toxicity, body weights, body weight gains, age of attainment of VO, body weight at VO, mean age at first estrus, mean cycle length, percent cycling, percent regular cycling, organ weights, serum T4 and TSH levels, or gross pathology.  Age and weight at VO were within the acceptable range of the performance criteria.
Chemical J was negative in the pubertal female assay. No treatment-related effects were seen for estrogen or thyroid-related endpoints when tested at doses up to and including 400 mg/kg/day.

     ix. Pubertal Male Assay (Rat)

In the male pubertal assay 16 Sprague-Dawley (Crl:CD[SD]) rats/dose group were treated daily via oral gavage with Chemical J in corn oil at doses of 0, 50, 200, or 400 mg/kg bw/day from post-natal day (PND) 23 to 53.  Animals were examined for preputial separation (PPS) daily beginning on PND 30, and weight at day of attainment was recorded.  Following sacrifice on PND 53, total serum testosterone, thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using radioimmunoassays (TSH) or chemiluminescent assays (testosterone and T4).  Liver, kidney, pituitary, adrenals, thyroid, and urogenital organ weights were recorded, and microscopic examinations of the thyroid, testes, epididymides, kidneys, and liver were performed.
As shown in Table 48, for Chemical J, at doses up to 400 mg/kg bw/day, no treatment-related effects were observed on mortality, clinical signs, body weight, body weight grain, weight at attainment of PPS, or serum T4 levels.  However, age at PPS was slightly increased (p<0.05) in the high dose group (PND 44.0 vs. PND 45.7).
As shown in Table 49, significant, dose-dependent decreases were also observed in the weights of the accessory sex tissues at doses of 200 mg/kg bw/day and 400 mg/kg bw/day.  At 200 mg/kg bw/day, weight decreases were noted in the seminal vesicle + coagulating gland (↓15-19%) and the ventral prostate (↓16%).  At 400 mg/kg bw/day, weight decreases were noted in the seminal vesicle + coagulating gland (↓33-41%), ventral prostate (↓27-29%), dorsolateral prostate (↓25-27%), and LABC complex (↓16-18%).  Concomitant with the weight decreases in these androgen sensitive tissues, there were significant, dose-dependent decreases in serum testosterone levels of 49% at 200 mg/kg bw/day and 85% at 400 mg/kg bw/day.
As shown in Table 50, other significant changes in organ weights included a 15% increase in relative adrenal weight at 400 mg/kg bw/day, and 8-10% decreases in pituitary weight at all dose levels that were not dose-dependent.  Other significant changes in clinical chemistry parameters observed at 200 and 400 mg/kg bw/day included:  21-32% increase in creatinine; 27% decreased in blood urea nitrogen; and a 17-21% decrease in potassium.  At 400 mg/kg bw/day, there were also significant changes in alkaline phosphatase (↓26%), total protein (↑4%) and chloride (↓3%).
No significant weight changes were noted in the thyroid, but increased thyroid follicular cell heights and decreased colloid area were noted at 400 mg/kg bw/day.   No changes were noted in serum T4 levels (Table 51).
Significant (p<0.05) increases were observed in liver weight (unadjusted and adjusted) and relative (to body) liver weight, which were dose-dependent.   Liver weights increased by 12-13% at 200 mg/kg bw/day and 27-28% at 400 mg/kg bw/day, and relative liver weights increased by 5%, 21% and 37% at 50, 200 and 400 mg/kg bw/day, respectively.  Concomitant with the increases in liver weight, there were minor histological changes at 200 and 400 mg/kg bw/day, which consisted of slight to very slight hypertrophy with altered tinctorial properties in centrilobular and midzonal hepatocytes (0/16 control, 16/16 at 200 mg/kg bw/day, and 15/15 at 400 mg/kg bw/day) and a very slight increase the numbers of mitotic figures in hepatocytes (2/15 control, 9/15 at 400 mg/kg bw/day).  A significant increase (53%) was also noted in gamma glutamyl transferase (GGT) at the high dose.

It appears the high dose (400 mg/kg bw/day) used in this assay is adequate based on the results from the assay that showed increased liver weights and microscopic liver findings. No treatment related changes were seen at the low dose (50 mg/kg bw/day).
At 200 mg/kg bw/day, delays in PPS, decreases in testosterone levels, changes in two androgen sensitive organs (seminal vesicle + coagulating gland and the ventral prostate) were reported. At 400 mg/kg bw/day, the observed changes were: delayed PPS, decreases in testosterone, decreases in four androgen sensitive tissues (seminal vesicle + coagulating gland, ventral prostate, dorsolateral prostate, and LABC complex).  Changes in the thyroid histopathology were also observed at 400 mg/kg bw/day along with a non-significant increasing trend in TSH across the treatment groups.
Chemical J was positive for the A (anti-A) pathway and negative for the T pathway in the pubertal male assay.

Table 48: Preputial Separation (PPS) in Male Rats Treated with Chemical J in the Male Pubertal Assay a
                              Parameter Evaluated
Vehicle Control
Chemical J
50 mg/kg bw/day
Chemical J
200 mg/kg bw/day
Chemical J
400 mg/kg bw/day

                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
Age at PPS (PND)
U
                                      15
                                     44.1
                                      1.7
                                      3.8
                                      16
                                     44.1
                                      1.4
                                      3.1
                                      16
                                     44.5
                                      1.6
                                      3.7
                                      15
                                     45.6
                                      2.9
                                      6.5

A
                                      15
                                     44.0
                                     0.44
                                      NA
                                      16
                                     44.1
                                     0.42
                                      NA
                                      16
                                     44.5
                                     0.42
                                      NA
                                      15
                                     45.7*
                                    (↑4)
                                     0.44
                                      NA
Proportion unseparated (#/N)
                                     0/15
                                     0/16
                                     0/16
                                     0/15
a Percent differences from controls, calculated by the reviewers, are included in parentheses.
U = Unadjusted for body weight on PND 23; A = Adjusted for body weight on PND 23; SD = Standard Deviation;
SE =Standard Error; used for means adjusted for body weight on PND 23
CV =Coefficient of Variation; NA=Not applicable
*Significantly different from controls at p<0.05.

Table 49:  Accessory Sex Organ Weights at Necropsy in Male Rats Treated with Chemical J in the Male Pubertal Assay [a]
                                     Organ
                                Vehicle Control
                                  Chemical J
                                50 mg/kg bw/day
                                  Chemical J
                               200 mg/kg bw/day
                                  Chemical J
                               400 mg/kg bw/day

                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
Seminal vesicle + coagulating gland, with fluid (mg)
U
                                      15
                                     522.2
                                     120.4
                                    23.1[b]
                                      16
                                     500.5
                                     115.3
                                      23
                                      16
                                    424.8*
                                    (↓19)
                                     116.1
                                     27.3
                                      15
                                    314.5*
                                    (↓40)
                                     109.2
                                     34.7

A
                                      15
                                     526.9
                                     28.0
                                      NA
                                      16
                                     500.5
                                     27.0
                                      NA
                                      16
                                    425.4*
                                    (↓19)
                                     27.0
                                      NA
                                      15
                                    309.2*
                                    (↓41)
                                     28.0
                                      NA
Seminal vesicle + coagulating gland, without fluid (mg)
U
                                      15
                                     280.2
                                     52.3
                                     18.7
                                      16
                                     275.7
                                     48.4
                                     17.6
                                      16
                                     240.8
                                     47.0
                                     19.5
                                      15
                                    188.2*
                                    (↓33)
                                     54.3
                                     28.8

A
                                      15
                                     282.3
                                     12.1
                                      NA
                                      16
                                     275.6
                                     11.7
                                      NA
                                      16
                                    241.1*
                                    (↓15)
                                     11.7
                                      NA
                                      15
                                    185.8*
                                    (↓34)
                                     12.1
                                      NA
Ventral prostate
(mg)
U
                                      15
                                     240.8
                                     34.2
                                     14.2
                                      16
                                     227.3
                                     35.0
                                     15.4
                                      16
                                    202.6*
                                    (↓16)
                                     42.4
                                     20.9
                                      14
                                    175.9*
                                    (↓27)
                                     38.2
                                     21.7

A
                                      15
                                     243.0
                                     8.76
                                      NA
                                      16
                                     227.6
                                     8.46
                                      NA
                                      16
                                    203.2*
                                    (↓16)
                                     8.46
                                      NA
                                      14
                                    172.5*
                                    (↓29)
                                     9.09
                                      NA
Dorsolateral prostate (mg)
U
                                      15
                                     156.1
                                     43.1
                                     27.6
                                      16
                                     147.2
                                     31.3
                                     21.3
                                      16
                                     143.6
                                     27.8
                                     19.4
                                      14
                                    117.3*
                                    (↓25)
                                     21.1
                                     18.0

A
                                      15
                                     157.8
                                     7.53
                                      NA
                                      16
                                     147.4
                                     7.27
                                      NA
                                      16
                                     144.1
                                     7.27
                                      NA
                                      14
                                    114.6*
                                    (↓27)
                                     7.81
                                      NA
LABC
(mg)
U
                                      15
                                     467.1
                                     90.5
                                     19.4
                                      16
                                     479.7
                                     68.2
                                     14.2
                                      16
                                     449.2
                                     68.4
                                     15.2
                                      15
                                    391.2*
                                    (↓16)
                                     73.2
                                     18.7

A
                                      15
                                     471.2
                                     17.1
                                      NA
                                      16
                                     479.6
                                     16.5
                                      NA
                                      16
                                     449.8
                                     16.5
                                      NA
                                      15
                                    386.6*
                                    (↓18)
                                     17.1
                                      NA
Epididymis, left (mg)
U
                                      15
                                      216
                                     23.6
                                     10.9
                                      16
                                      219
                                     35.8
                                     16.3
                                      16
                                     206.8
                                     20.5
                                      9.9
                                      15
                                     202.3
                                     27.5
                                     13.6

A
                                      15
                                     217.3
                                     6.47
                                      NA
                                      16
                                     218.9
                                     6.25
                                      NA
                                      16
                                     207.0
                                     6.25
                                      NA
                                      15
                                     200.9
                                     6.47
                                      NA
Epididymis, right (mg)
U
                                      15
                                     213.4
                                     20.7
                                      9.7
                                      16
                                     221.6
                                     25.9
                                     11.7
                                      16
                                     213.4
                                     29.4
                                     13.8
                                      15
                                     207.4
                                     26.9
                                     13.0

A
                                      15
                                     214.3
                                     6.44
                                      NA
                                      16
                                     221.5
                                     6.23
                                      NA
                                      16
                                     213.5
                                     6.23
                                      NA
                                      15
                                     206.4
                                     6.44
                                      NA
Testis, left (mg)
U
                                      15
                                     1412
                                     109.3
                                      7.7
                                      16
                                     1429
                                     142.7
                                      10
                                      16
                                     1417
                                     115.4
                                      8.1
                                      15
                                     1440
                                     136.3
                                      9.5

A
                                      15
                                     1415
                                     32.5
                                      NA
                                      16
                                     1429
                                     31.5
                                      NA
                                      16
                                     1417
                                     31.5
                                      NA
                                      15
                                     1437
                                     32.6
                                      NA
Testis, right (mg)
U
                                      15
                                     1423
                                     119.4
                                      8.4
                                      16
                                     1448
                                     138.1
                                      9.5
                                      16
                                     1428
                                     85.3
                                      6.0
                                      15
                                     1451
                                     128.3
                                      8.8

A
                                      15
                                     1426
                                     30.3
                                      NA
                                      16
                                     1448
                                     29.3
                                      NA
                                      16
                                     1428
                                     29.3
                                      NA
                                      15
                                     1447
                                     30.3
                                      NA
a Percent differences from controls, calculated by the reviewers, are included in parentheses.
b The %CV exceeds the maximum acceptable %CV.
U =Unadjusted for body weight on PND 23;
A=Adjusted for body weight on PND 23;
R=Organ-to-body weight ratio (relative to body weight);
SD=Standard Deviation;
SE =Standard Error; used for means adjusted for body weight on PND 23; CV=	Coefficient of Variation
*Significantly different from controls at p<0.05.
Table 50:  Additional Organ Weights at Necropsy in Male Rats Treated with Chemical J in the Male Pubertal Assay [a]
                                     Organ
                                Vehicle Control
                                  Chemical J
                                50 mg/kg bw/day
                                  Chemical J
                               200 mg/kg bw/day
                                  Chemical J
                               400 mg/kg bw/day

                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD/
                                      SE
                                      CV
                                      (%)
Liver
(g)
U
                                      15
                                     13.1
                                     1.77
                                     13.5
                                      16
                                     13.2
                                     1.22
                                     9.25
                                      16
                                     14.8*
                                    (↑13)
                                     1.24
                                     8.35
                                      15
                                     16.8*
                                    (↑28)
                                     1.64
                                     9.79

A
                                      15
                                     13.2
                                     0.36
                                      NA
                                      16
                                     13.2
                                     0.343
                                      NA
                                      16
                                     14.8*
                                    (↑12)
                                     0.343
                                      NA
                                      15
                                     16.7*
                                    (↑27)
                                     0.355
                                      NA

R
                                      15
                                     4.46
                                     0.22
                                     4.97
                                      16
                                     4.67*
                                    (↑5)
                                     0.21
                                     4.52
                                      16
                                     5.41*
                                    (↑21)
                                     0.23
                                     4.33
                                      15
                                 6.09* (↑37)
                                     0.23
                                     3.83
Pituitary
(mg)
U
                                      15
                                      9.6
                                      0.9
                                      9.5
                                      16
                                     8.8*
                                    (↓8)
                                      0.7
                                      8.1
                                      16
                                     8.6*
                                    (↓10)
                                      0.9
                                     10.0
                                      15
                                     8.6*
                                    (↓10)
                                      1.1
                                     12.3

A
                                      15
                                      9.6
                                     0.23
                                      NA
                                      16
                                     8.8*
                                    (↓8)
                                     0.22
                                      NA
                                      16
                                     8.6*
                                    (↓10)
                                     0.22
                                      NA
                                      15
                                     8.6*
                                    (↓10)
                                     0.23
                                      NA

R
                                      15
                                      3.3
                                      0.3
                                      9.0
                                      16
                                      3.1
                                      0.2
                                      7.4
                                      16
                                      3.2
                                      0.3
                                     10.1
                                      15
                                      3.2
                                      0.3
                                     10.9
Adrenals
(mg)
U
                                      15
                                     43.2
                                      7.4
                                      17
                                      16
                                      44
                                      6.2
                                     14.1
                                      16
                                     43.4
                                      7.8
                                     17.9
                                      15
                                     46.8
                                      7.2
                                     15.3

A
                                      15
                                     43.5
                                     1.69
                                      NA
                                      16
                                     44.0
                                     1.64
                                      NA
                                      16
                                     43.4
                                     1.64
                                      NA
                                      15
                                     46.4
                                      1.7
                                      NA

R
                                      15
                                     14.8
                                      2.2
                                     14.6
                                      16
                                     15.6
                                      1.8
                                     11.2
                                      16
                                     15.9
                                      2.5
                                      16
                                      15
                                      17*
                                    (↑15)
                                       2
                                     11.7
Thyroid, fixed
(mg)
U
                                      15
                                    11.3[b]
                                      1.5
                                     13.6
                                      16
                                     11.9
                                      1.8
                                     15.4
                                      15
                                     12.5
                                      1.3
                                     10.6
                                      15
                                     12.6
                                      1.5
                                     11.9

A
                                      15
                                     11.3
                                     0.41
                                      NA
                                      16
                                     11.9
                                     0.39
                                      NA
                                      15
                                     12.5
                                     0.41
                                      NA
                                      15
                                     12.6
                                     0.41
                                      NA
a Percent differences from controls, calculated by the reviewers, are included in parentheses.
b The mean weight was less than the acceptable weight range.
c The %CV exceeds the maximum acceptable %CV.
U = Unadjusted for body weight on PND 23;
A=Adjusted for body weight on PND 23;
R=Organ-to-body weight ratio (relative to body weight);
SD=Standard Deviation; SE = Standard Error; used for means adjusted for body weight on PND 23;
CV = Coefficient of Variation
*Significantly different from controls at p<0.05.

Table 51:  Hormone Levels in Male Rats Treated with Chemical J in the Male Pubertal Assay [a]
                              Parameter Evaluated
                                Vehicle Control
                                  Chemical J
                                50 mg/kg bw/day
                                  Chemical J
                               200 mg/kg bw/day
                                  Chemical J
                               400 mg/kg bw/day

                                  # of Males
                                     Mean
                                      SD
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD
                                      CV
                                      (%)
                                  # of Males
                                     Mean
                                      SD
                                      CV
                                      (%)
                                   Hormones
Serum T4, Total (ug/dL)
                                      15
                                     4.78
                                     0.670
                                     14.0
                                      16
                                     4.69
                                     0.481
                                     10.3
                                      16
                                     5.19
                                     0.724
                                     14.0
                                      15
                                     5.15
                                     0.458
                                     8.89
Serum TSH (ng/mL)
                                      15
                                     7.07
                                     3.23
                                     45.7
                                      16
                                     8.74
                                     6.10
                                     69.7
                                      16
                                     9.06
                                     5.14
                                     56.8
                                      15
                                     11.5
                                     6.52
                                     58.5
Serum
testosterone (ng/mL)
                                      15
                                     3.62
                                     2.36
                                     65.1
                                   (45.7)[b]
                                      16
                                     3.46
                                     3.24
                                     93.7
                                      16
                                     1.83*
                                    (↓49)
                                     1.31
                                     71.5
                                      15
                                 0.561*(↓85)
                                     0.468
                                     83.6
a Percent differences from controls, calculated by the reviewers, are included in parentheses
b When animal #5998 is excluded from data set (in parentheses) the %CV is less.
SD = Standard Deviation;
CV= Coefficient of Variation
*Significantly different from controls at p<0.05.

     x.  Fish Short-term Reproduction Assay (FSTRA)
The 21-day fish short-term reproduction assay of Chemical J with fathead minnows (Pimephales promelas) was studied under flow-through conditions.  Adult fish, 16 spawning groups (2 males and 4 females in each group; ca. 5.75 months old) were exposed to Chemical J at nominal concentrations of 0 (control), 0.032 0.32, and 3.2 mg a.i./L; time-weighted average-measured concentrations were <LOQ (<0.00510), 0.0338, 0.327, and 3.30 mg a.i./L.  The test system was maintained at 24.6 to 25.4[o]C and a pH of 7.2 to 7.5.  All performance criteria were met in this study.
Spawning frequency in the negative control occurred at least every 4 days and fecundity ranged from 21 to 33 eggs/female/day/replicate. Fecundity was 25.6, 20.1, 22.6 and 0.550 eggs/female/day/replicate in the 0 (control), 0.0338 0.327, and 3.3 mg ai/L treatment levels, respectively.  Fecundity at the 3.3 mg a.i./L level was 98% lower than the control. No significant effect was detected for fertilization success. Fertilization rate in the control was 99%, compared to 99.5, 99.1% and 100% in the 0.0338, 0.327 and 3.3 mg a.i./L treatment groups.  Female gonadosomatic index (GSI) exhibited a significant (p<0.05) increase of 41 and 46% at the 0.327 and 3.3 mg a.i./L levels, respectively, while male GSI was significantly (p<0.05) increased 79% at the 3.3 mg a.i./L level.
Female plasma vitellogenin (VTG) was significantly reduced (p<0.05) by 88% relative to controls at the 3.3 mg a.i./L level, while no effect was detected for male plasma VTG. Additionally, there was a significant 200% increase (p<0.05) in female testosterone levels at the 0.327 mg a.i./L level, relative to the control.  While not statistically significant (p=0.067), a 85% reduction in female plasma 17-estradiol concentrations at the 3.3 mg ai/L level was observed which was considered biologically significant. Histopathology on female gonads reported severe oocyte atresia (as reported by study pathologist using procedures described in guideline) at the 3.3 mg a.i./L level relative to the control . Male fish gonads were reported as having a increased proportions of mature sperm (gonadal stage 3) in the 3.3 mg ai/L treatment, relative to the control.
There were no significant effects on mortality (p>0.05).  Minimal effects on secondary sex characteristics were observed in the study with one male fish in the 3.3 mg a.i./L and in the control were reported to have very thin fatpads while one fish each in the low and high treatment were reported to exhibit loss of territorial aggressiveness. These observations did not appear to be treatment-related as they were reported in both the control and treatment groups. Additionally, a caudal fin was reported as missing in one female fish at the mid-treatment group.  In the 3.3 mg a.i./L level, a significant reduction (9%) in female body weight was observed (using a statistical trend test); however, the impact of this magnitude of reduction, in this assay, is not considered significant. The results are presented in Table 52 through Table 54.
In summary, at the highest treatment group, i.e., 3.3 mg a.i./L, there was a significant (p<0.05) decrease in fecundity and female plasma VTG and estradiol, along with a significant increase in male and female GSI. Additionally, alterations in male and female gonadal histopathology were reported at the highest treatment group.  Furthermore, a significant increase in female GSI and plasma testosterone was reported at 0.32 mg a.i./L.  While a significant (p<0.05) decrease in female body weight was observed at 3.3 mg a.i./L, given the magnitude of effect (9% decrease), the impact on the interpretation of the results of this assay is not considered to be significant.
Given the reported effects and the complementarity among the responses (Vtg, GSI, fecundity, T and E), Chemical J is considered to be positive for potential interactions with the E and A pathways.
Table 52:  Chemical J - Reproductive and HPG Endpoints for Male Pimephales promelas in the FSTRA

                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                Tubercle Score
                                      GSI
                     Gonadal Staging and Histopathology[1]
                                  Plasma VTG
                                   Plasma T
                                   Plasma E2

                                    Median
                                 % Difference
                               Effect? (Yes/No)
                                 % Difference
                                 % Difference
                                 % Difference
Control (<LOQ)
                                     18.3
                                       0
                                      NA
                                       0
                                       0
                                       0
0.032 (0.0338)
                                     20.5
                                     16.50
                                      No
                                     -1.40
                                    -40.97
                                    -31.08
0.32 (0.327)
                                     19.3
                                     27.30
                                      No
                                    -28.01
                                     40.97
                                     10.45
3.2 (3.3)
                                     15.5
                                   78.99[2]
                                      Yes
                                    -83.06
                                    -31.02
                                    -18.82
Abbreviations:.   [E2] 17β-estradiol.  [GSI] Gonado-Somatic Index.  [NA] Not applicable.
  [T] Testosterone.  [VTG] Vitellogenin.
[1]  Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.
[2]  Statistically significant  [p<0.05].
LOQ=<0.00510 mg ai/L

Table 53.  Chemical J -Reproductive and HPG Endpoints for Female Pimephales promelas in the FSTRA

Treatment
(mg a.i../L)
[measured]
Fecundity
Fertility Success
Tubercle Score
GSI
Gonadal Staging and Histopathology[1]
Plasma VTG
                                   Plasma T
                                   Plasma E2

                                   % Diffe.
                                    % Diff.
                                    Median
                                    % Diff.
                               Effect? (Yes/No)
                                    % Diff.
                                    % Diff.
                                    % Diff.
Control (<LOQ)
                                      NA
                                      NA
                                       0
                                      NA
                                      NA
                                      NA
                                      NA
                                      NA
0.032 (0.0338)
                                     -21.7
                                     0.45
                                       0
                                     22.8
                                      No
                                     9.16
                                     74.0
                                     -33.7
0.32 (0.327)
                                     -11.8
                                     0.00
                                       0
                                    40.6[2]
                                      No
                                     -33.5
                                    201[2]
                                     13.7
3.2 (3.30)
                                   -97.9[2]
                                     0.96
                                       0
                                    46.2[2]
                                      Yes
                                   -88.0[2]
                                     14.4
                                   -84.8[2]
Abbreviations:  Diff. Difference.  [E2] 17β-estradiol.  [Fert.] Fertilization.  [GSI] Gonado-Somatic Index.
  [NA] Not applicable.  [T]Testosterone.  [VTG] Vitellogenin.
[1]  Conclusions regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.
[2]  Statistically significant at p<0.05.
LOQ=<0.00510 mg ai/L

Table 54: Chemical J - Growth Endpoints in the Fish Short-Term Reproduction Assay (FSTRA)
                                   Treatment
                                  (mg a.i./L)
                                  [measured]
                                  Body Weight
                                    Length

                                     Males
                                    Females
                                     Males
                                    Females

                                 % Difference
                                 % Difference
                                 % Difference
                                 % Difference
Control (<LOQ)
                                      NA
                                      NA
                                      NA
                                      NA
0.032 (0.338)
                                     21.53
                                     -2.09
                                     6.52
                                     -1.30
0.32 (0.327)
                                     7.49
                                     -1.61
                                     0.20
                                     -2.60
3.2 (3.30)
                                     4.79
                                   -8.86[1]
                                     4.49
                                     -2.78
	Abbreviations:  [NA] Not applicable.
	1  Statistically significant using a trend test (Jonckheere-Terpstra) [p<0.05].
	LOQ=<0.00510 mg ai/L

 xi.  Amphibian Metamorphosis Assay (AMA)
The 21-day assay of Chemical J on amphibian metamorphosis of African clawed frog (Xenopus laevis) was studied under flow-through conditions.  Amphibian larvae (80 per control and treatment group, NF stage 51) were exposed to a negative control and nominal concentrations of 0.100, 0.500, and 2.50 mg a.i./L. Time-weighted average (TWA) concentrations were <0.00474 (<LOQ; control), 0.0964, 0.438, and 2.04 mg a.i./L. The test system was maintained at 20.1 to 22.9°C and a pH of 7.0 to 7.7.
Chemical J significantly reduced wet weight in the two highest treatment groups on Day 7 (>=38%, p <0.05) and in the lowest and highest treatment groups on Day 21 (>=20%, p<0.01). Similarly, snout-vent length (SVL) was significantly reduced at the two highest treatment groups on Day 7 (>=16%, p <= 0.05) and significant reductions were detected in the lowest and highest treatment groups on Day 21 (6%, p <= 0.01). There were also significant reductions in non-normalized hind limb length (HLL) at the two highest treatment groups on Day 7 (>=20%, p < 0.05) and at all treatment groups on Day 21 (>=15%, p <= 0.01).
Statistically significant developmental delays (p<0.05) were observed at Chemical J concentrations associated with growth inhibition and clinical signs of toxicity.   On Day 7, developmental stage was significantly (p<0.05) delayed at the highest treatment level. Normalized HLL on Day 7 was significantly decreased at the highest treatment level (13%, p= 0.025 at 2.04 mg a.i./L) and was significantly decreased at all treatment levels on Day 21 (10%, 24%, 23% at the 0.0964, 0.438, and 2.04 mg a.i./L levels, respectively, p <0.05).  There was no evidence of asynchronous development reported on Days 7 or 21. There were no treatment-related effects on thyroid gland histopathology at any treatment level.
Some transient abnormal behaviors (floating on the surface, lying on the bottom of the tank, inverted or irregular swimming, lack of surfacing activity, and being nonresponsive to stimulus) were noted among control and treated tadpoles, with a greater proportion of these abnormal behaviors observed among those in the two highest treatment groups. There was no mortality in the control group and mortality did not exceed 7% in any treatment level. All validity requirements for the amphibian metamorphosis assay were met. The performance criteria were not fully met because there were not >=2 concentrations (non-control) without overt toxicity available for analysis.  Additionally, there was a temperature fluctuation (i.e., 20.1°C) on Day 1 in the continuously-monitored control test vessel.
The presence of clinical signs of toxicity and inhibition of growth at the same concentrations as developmental delays, in the absence of thyroid histopathological effects, makes it difficult to interpret whether Chemical J has the potential to interact with the hypothalamic-pituitary-thyroid (HPT) axis within the range of concentrations tested in the AMA. However, based on the results in the lowest treatment group which reported no observed clinical signs of toxicity, Chemical J is considered not likely to interact with the thyroid pathway in the AMA.
Other Scientifically Relevant Information (OSRI) for Chemical J
The registrant elected to conduct the 11 Tier 1 screening assays, therefore, no OSRI were submitted to the agency. However, Chemical J has Part 158 mammalian and ecotoxicity studies previously submitted to the Agency. These studies are discussed below.
      1.  Mammalian OSRI Data
In a 3-month feeding study, groups of 10 male and 10 female COBS-CD (SD) BR rats were administered technical Chemical J in the diet at 0, 0.25, 0.75, 2.5, 7.5, 75, 250 or 750 mg/kg bw-day. During weeks 3 and 4 dietary exposure levels, were increased to 0.35, 1.05, 3.5, 10.5, 35, 105, 350 or 1050 mg/kg bw-day. During weeks 5-13, dietary exposure levels were again increased to 10, 30, 100, 300, 1000, 3000, 10,000 or 30,000 ppm (0.5, 1.5, 5, 15, 50, 150, 500 or 1500 mg/kg bw-day). Evaluations included, but were not limited to, clinical observations and mortality, body weight, food consumption, hematology, clinical chemistry, organ weights, gross necropsy and histology. Complete histological examinations were performed in the 0, 150 and 500 mg/kg bw-day groups. All rats treated at 1500 mg/kg bw-day died during the dosing period; no treatment-related deaths occurred at lower doses. At 150 mg/kg bw-day and above, an increase incidence in small follicles was observed in the thyroid for males.  Additionally, effects on the liver and kidneys were observed (i.e., changes in weights and histopathology) at the 150 mg/kg bw-day dose.

In a 1-year feeding study, groups of 6 male and 6 female Beagle dogs were administered technical Chemical J at dietary concentrations of 0, 10, 100, 400 or 1600 ppm. Based on measured food consumption and body weight, the average chemical intakes were 0, 0.34, 3.09, 14.28 and 54.22 mg/kg bw-day in males and 0, 0.4, 3.83, 15.68 and 58.2 mg/kg bw-day in females. Endpoints reported included body weight, food and water consumption, hematology, clinical chemistry, organ weights and histopathology. Complete histological examinations were not reported on the low and mid-dose animals; however, assessment in all dietary treatment groups included testes and liver. No effects on thyroid or pituitary weights were reported.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, thyroid or pituitary compared to the control group.  Liver effects including, hepatocellular hypertrophy and increased liver weight were observed 14.28 and 15.68 mg/kg bw- day for males and females, respectively.

In a 2-year chronic/carcinogenicity study, Chemical J was administered to 110 male and 110 female Sprague Dawley rats in the diet.  Dietary levels for the low-dose group were 25/35/50 ppm; for the mid-dose group were 100/140/200 ppm and for the high-dose group were 400/560/800 ppm.   The first dose level in each series was administered for a period of 2 weeks, the second dose level in each series was administered for a period of 2 weeks and the third dose level in each series was administered from weeks 5 to termination.  The overall calculated mean daily compound consumption was 0, 2.49, 9.84 or 39.21 mg/kg bw/day for males and 0, 3.23, 12.86 or 52.34 mg/kg bw/day for females.  Evaluations included clinical observations and mortality, body weight, food and water consumption, hematology, clinical chemistry, gross necropsy and histopathology.  At 9.84 mg/kg bw/day in males, the mean absolute testes weights were significantly less than controls (p<0.05; 77% of controls) at study termination.  An increase in testicular atrophy was observed at 39.21 mg/kg bw/day.  Microscopically, the seminiferous tubules were frequently devoid of spermatid formation and germinal epithelial cells at the highest dose tested. No adverse histopathological effects were seen in the prostate, ovaries, uterus, mammary gland, pituitary, or thyroid at any dose tested.   No changes in ovary or thyroid weights were reported.

In a 2-year supplemental carcinogenicity study, Chemical J was administered to 50 male and 50 female Sprague Dawley rats at dose levels of 0 or 2500 ppm (125 mg/kg bw/day) in the diet.  Evaluations included clinical observations and mortality, body weight, food and water consumption, hematology, clinical chemistry, gross necropsy and histopathology. At 125 mg/kg bw/day, Absolute and relative testes weights were significantly lower in treated males at both the interim sacrifice (63% and 68% of controls for right testes, 58% and 63% of controls for left testes) and terminal sacrifice (81% and 85% of controls for right testes, and 73% and 74% of controls for left testes).  An increase in bilateral aspermatogenesis was also observed at this dose.  The decreased spermatogenic activity was associated with an increase incidence of hypospermia and cellular debris in the epididymis of treated males.  Liver effects including hypertrophy and vacuolization were also observed at 125 mg/kg bw/day.

In a 2-year feeding/oncogenicity study, groups of 110 male and 110 female Crl:CD-1(ICR)BR mice (Mus musculus) were administered technical Chemical J at dietary concentrations of 0, 20 , 100 or 500 ppm. The overall reported mean daily compound consumption was 0, 2.7, 13.7 or 70.2 mg/kg bw- day for males and 0, 3.2, 16.5 or 85.2 mg/kg bw-day for females. Of the 110 mice/sex/dose, 10/sex/dose were euthanized at 3 and 6 months and 20/sex/dose group were euthanized at 12 months. A sentinel animal program was also conducted separately in which 25 mice/sex were evaluated at 24 months. Evaluations included, but was not limited to, clinical observations and mortality, body weight, food and water consumption, clinical chemistry, gross necropsy and histopathology. No effects on the weights of the thyroid, ovaries or pituitary were observed.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, mammary, thyroid or pituitary compared to the controls. Effects on the liver (increases in liver enzyme (SGPT), liver weight and hepatic histopathology in one or both sexes) were observed at 500 mg/kg diet (70.2 mg/kg bw-day for males and 85.2 mg/kg bw-day for females).

In an 18-month feeding/oncogenicity study, groups of 60 female Crl:CD-1 (ICR)BR mice were administered technical Chemical J at dietary concentrations of 0 or 2000 ppm (393.5 mg/kg bw-day). Ten mice/group were euthanized at 52 weeks. An additional 20 mice/group were added to the study as sentinels and also euthanized at 52 weeks. This study was conducted at a dietary level approaching the maximum tolerated level because it was determined that a previous study was not conducted at sufficiently high dietary treatment levels in females. Evaluations included clinical observations and mortality, body weight, food and water consumption, hematology, gross necropsy and histopathology. Clinical chemistry and urine analyses were not performed.
No effects on the weights of the thyroid, ovaries or pituitary were observed.  There were no histological changes in the testes, seminal vesicles, prostate, ovary, uterus, mammary, thyroid or pituitary compared to the controls.  Effects on body and liver weight, and liver and adrenal histopathology were observed at 393.5 mg/kg bw-day, the lowest concentration tested.
In a prenatal developmental toxicity study in Sprague-Dawley rats, Chemical J in corn oil was administered at doses of 0, 31.3, 93.8, 312.6 or 468.9 mg/kg bw/day by gavage on gestation days 6-15.  For maternal toxicity, the NOAEL was 93.8 mg/kg bw/day and the LOAEL was 312.6 mg/kg bw/day based on clinical signs (rough hair coat, salivation, alopecia, desquamation and red exudate around the mouth).  The developmental toxicity NOAEL was 93.8 mg/kg bw/day and the developmental toxicity LOAEL was 312.6 mg/kg bw/day, based on increased incidences of 14[th] rudimentary ribs and 7[th] cervical ribs.  In a range-finding study, 100% maternal mortality was seen at 700 mg/kg bw/day.
A prenatal developmental toxicity study was conducted in pregnant New Zealand white rabbits, in which Chemical J (in 1% methylcellulose) was administered by gavage at doses of 0, 20, 60, or 200 mg/kg bw/day on gestation days 7-19. For maternal toxicity, the NOAEL was 60 mg/kg bw/day and the LOAEL was 200 mg/kg bw/day, based on decreased body weight and body weight gain during dosing, with a rebound weight gain effect during the post-treatment period, and on observations of irregular feces, bloody urine, bloody urogenital or anal area, and blood and/or aborted materials in the cage pan (total litter loss occurred in three does).  For developmental toxicity the NOAEL was 60 mg/kg bw/day and the LOAEL was 200 mg/kg bw/day based on increased resorptions, decreased litter size, and decreased viability index.   In a range-findings study, maternal mortality was observed at doses of 464 and 700 mg/kg bw/day, and the maternal and developmental toxicity NOAELs for that study were 100 mg/kg bw/day.
In a  reproduction study, male and female CRL:CD(SD)BR rats were fed diets containing Chemical J at 0, 50, 200 or 1000 ppm (2.5, 10 or 50 mg/kg bw/day) for two consecutive generations.  At 200 ppm, centrilobular hepatocellular hypertrophy was observed in the P2 males.  This was supported by slight but statistically significant increases in liver weights in males.  At 1000 ppm, centrilobular hepatocellular hypertrophy was observed in both sexes in the P1 and P2 generations.  These were again supported by slight but statistically significant increases in liver weights.  For parental systemic toxicity, the NOAEL was 50 ppm (2.5 mg/kg bw/day) and the LOAEL was 200 ppm (10 mg/kg bw/day) based on hepatocellular hypertrophy and increases in liver weights.  At 1000 ppm, an increase in the number of stillborn or percent born dead pups was observed in both generations.  In addition, multifocal or diffuse testicular atrophy was observed in males in the P2 generation.  Increased necrotic spermatocytes/ spermatids or decreased spermatozoa and atrophy of the prostate were also observed in these animals. Sexual maturation, estrous cycle length and periodicity, and sperm parameters were not evaluated in this study.   For reproductive toxicity, the NOAEL was 200 ppm (10 mg/kg bw/day) and the LOAEL was 1000 ppm (50 mg/kg bw/day) based on an increased incidence in the number of stillborn pups and atrophy of the testes and prostate.  At 1000 ppm, there was a slight decrease in pup weight gain during lactation.  For developmental toxicity, the NOAEL was 200 ppm (10 mg/kg bw/day) and the LOAEL was 1000 ppm (50 mg/kg bw/day) based on a decrease in pup body weight gain during lactation.
In evaluating the available mammalian Part 158 data for Chemical J for endpoints relevant to the E, A, or T pathway, effects on testes and prostate were observed in the two-generation reproduction rat Part 158 study,( multifocal or diffuse testicular atrophy, increased necrotic spermatocytes/spermatids or decreased spermatozoa and atrophy of the prostate).  Additionally, decreased testes weight, testicular atrophy and effects on spermatogenesis were observed in the rat chronic/carcinogenicity studies.

2.  Ecotoxicity OSRI Data
In an early life-cycle toxicity test with fathead minnow (Pimephales promelas) after 35 days of exposure to Chemical J, the NOEC was reported at 0.98 mg /L, with a LOEC of 2.2 mg/L, based on reduction in standard length compared to the control.  Wet weight was affected at 4 mg/L.
On an acute exposure basis, Chemical J is classified as "moderately toxic" to freshwater and estuarine/marine fish with a 96-hour LC50 of 2.4 mg a.i./L in bluegill sunfish (Lepomis macrochirus) and 4.7 mg a.i./L in sheepshead minnow.
In a one-generation reproduction study with mallard ducks  and Northern bobwhite quail with Chemical J, no effects on adult birds, hatchlings or reproductive parameters was observed at any concentration tested. The NOAEC was 260 mg/kg-diet, the highest concentration tested.
In a one-generation reproduction study with mallard ducks and Northern bobwhite quail with Chemical J, no effects on reproduction were observed at any concentration tested. The NOAEC was 60 mg/kg-diet, the highest concentration tested.
In a chronic 28-day toxicity test with the mysid shrimp exposure to Chemical J at concentrations between 2.58 and 85.6 μg ai/L did not cause any significant decreases on F0 survival, F0 male or female body length, number of young produced per female, survival of F1 mysids, or F1 male or female body length. No significant increase was observed in time to first brood.  However, there was a significant decrease (p<0.050 in time to first brood at intermediate test concentrations between 5.09 and 42.0 ug a.i./L, but not at the highest concentration of 85.6 ug a.i./L. At each of the middle test concentrations, the mean time to first brood was approximately one day less than the control.
No additional data with frog or other amphibian species have been submitted.
In evaluating the available ecotoxicity Part 158 data for Chemical J for endpoints relevant to the E, A or T pathway, reproduction did not appear to be altered (birds and mysids), except for a significant decrease in the time to first brood compared to the control in the chronic mysid shrimp study.

Discussion

          ii. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis
As previously noted, the EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical J has potential to interact with the HPG axis or its downstream pathways related to steroidogenesis or sex steroid signaling.  The Tier 1 in vitro assays relevant to this analysis include the AR, binding, ER binding, ERTA, aromatase, and steroidogenesis assay.  Relevant in vivo assays include the male and female pubertal rat, uterotrophic, Hershberger, and the FSTRA assays.

Effects on Estrogen Pathway: Table 5 below summarizes the results of the EDSP Tier 1 screening assay results and OSRI for determining the potential for Chemical J to interact with the estrogen pathway.  The various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic, or HPG axis effects.

Chemical J was negative for both the in vitro ER binding assay and ERTA assay suggesting that Chemical J does not trigger a molecular initiating event (MIE) with signaling through the estrogen receptor.  Furthermore, a negative response was observed in the uterotrophic assay, supporting this assertion.  However, Chemical J was positive for alterations in enzyme activity in both the in vitro steroidogenesis assay and aromatase assay, suggesting that Chemical J may alter hormone production along the steroidogenesis pathway.  In the in vitro steroidogenesis assay, significant decreases (p<0.05) in estrogen production were reported. Moreover, in the in vitro aromatase assay, Chemical J was shown to strongly inhibit aromatase activity (i.e., conversion of androgen to estrogen).

These observed alterations to steroidogenesis which would result in decrease estrogen levels are corroborated by the reported results in the FSTRA in which alterations to several complementary endpoints were observed.  In the FSTRA at the highest concentration tested, a significant decrease (p<0.05) in estradiol (↓85% )in female fish was reported with a decrease in VTG (↓88%) along with an significant increase in GSI (↑46%) and significant decrease in the apical endpoint of fecundity (↓98%).  An increase in gonadal oocyte atresia in females was also observed at this high concentration.  Additionally, a significant increase in male GSI (↑80%) along with alterations in gonadal histopathology was reported.