Chemical A did not significantly affect 7-day normalized hind-limb length (HLL) at 0.0076, 0.082 and 0.820 mg a.i./L.  Reductions in normalized HLL at day 21 in the 0.082 and 0.820 mg a.i./L treatment levels were similar, based on comparison to the negative control, but the effect was only statistically significant at the intermediate treatment level (p=0.045).   No significant change of median Nieuwkoop-Faber (NF) developmental stage was observed at 7 days or at 21 days at any treatment level. Asynchronous development was not observed in any of the treatments.   Body weight was significantly reduced on day 21 at the highest treatment level only (p=0.01); no other  effects on snout-vent length (SVL) or body weight were observed.  Thyroid gland hypertrophy was observed in the 0.820 mg/L treatment.  With respect to thyroid histopathology, mild follicular cell hypertrophy and hyperplasia were reported as common in both control and treatment specimens. Incidence of increased or decreased follicular lumen area was sporadic but again did not appear to be treatment-related. Clinical signs including discoloration and curvature of the spine (incidence values not reported) were observed in several tadpoles at each treatment concentration and in controls. Unless otherwise indicated, all effects are reported based on comparison to the negative control, and late stage tadpoles (NF Stage > 60) are excluded from analyses of continuous endpoints (SVL, body weight, and normalized HLL) due to the effects of normal tissue resorption during metamorphosis.
The absence of significant effects on thyroid-related endpoints in the AMA suggests that Chemical A did not interact with the amphibian HPT axis or its downstream thyroid hormonal pathway under these study conditions.

Other Scientifically Relevant Information (OSRI) for Chemical A
This section of the case study provides a summary of the Agency review of existing data cited as OSRI.  For purposes of this case study, the information described below only includes data submitted by the test order recipient or the public and/or data submitted to support pesticide registration (e.g., Part 158 guideline studies).  Existing data includes data previously submitted to the Agency in support of a registration decision believed to be relevant to one or more of the Tier 1 assays.
                 1.         In Vitro OSRI Data
Kojima et al. (2005) investigated the response of an in vitro estrogen receptor - mediated reporter gene assay following exposure to 32 chemical pesticides either alone or in combination.  The E-CALUX[(R)] Assay (Xenobiotic Detection Systems, Inc, NC, USA) uses a human ovarian carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a mouse mammary tumor virus promoter.  Estrogen-responsive concatemers (i.e., repetitive DNA sequences) are inserted directly above the promoter confer estrogen responsiveness to the viral promoter and reporter gene.  The transfected cells are made stable by an antibiotic resistance gene resulting in the cell line used in the assay (BG1Luc4E2).  Chemicals that interact with estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms of Relative Light Units (RLU).
The assays were conducted by growing the BG1Luc4E2 cells in medium for four days, transferring 2 x 10[5] cells/mL into 96-well plates and incubating at 37˚C with 5% CO2 for 24 h.  The medium was replaced, each test compound or an estrogen standard (E2) was added, and the plates were incubated at 37˚C with 5% CO2 for 24 h.  The medium was replaced and the cells were lysed with Promega lyse buffer.  Luciferase activity was measured via chemiluminescence with a Berthold[(R)] Microplate Luminometer in order to compare treatment RLU to an E2 standard.  For comparative purposes, the EC10 was used (Effective Concentration at which 10% of the maximum response was observed).  The data were not sufficient to allow calculation of an median effect concentration (EC50) at which 50% of the maximum response was observed.  The EC10 of E2 was 3.2 x10-12 M, which is within the expected range, and the dose response curve in the paper indicates that the assay performed as expected for chemicals that have positive estrogenic activity.
Chemical A was shown to have some estrogenic activity in the BG1Luc4E2 assay with a reported EC10 of 4.6 x10[-4] M, which is 8 orders of magnitude lower than the E2 standard.
                 2. Mammalian OSRI Data
In a subchronic dietary toxicity study, male and female Sprague-Dawley rats 5 months) were fed diets containing Chemical A at levels equivalent to  0, 0.03, 0.3, 15, and 168 mg/kg/day for males and 0, 0.04, 0.4, 19 and 212 mg/kg/day for females in the diet for 13 weeks. No treatment-related changes were seen either in the absolute or relative weights of the testes, epididymides, prostate, uterus, ovaries, thyroid, adrenal or the pituitary glands. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, ovaries, adrenal, thyroid or the pituitary glands. Clinical signs (hypersensitivity to sound and touch; hyperactivity and aggressiveness) were only observed at the highest dose.
In a chronic dietary toxicity study, male and female Beagle dogs  were fed diets containing Chemical A at 0, 0.0032, 0.015, 4.7 and 7.7 mg/kg/day for males and 0, 0.0037, 0.020, 4.5 and 9.1 mg/kg/day for females in the diet for 52 weeks. No treatment-related changes were seen either in the absolute or relative weights of the testes, epididymides, prostate, uterus, ovaries, or the adrenal and thyroid glands.  No treatment-related histopathological lesions were seen in the testes, epididymides, prostate, uterus, ovaries, vagina, mammary, adrenal, thyroid or the pituitary glands
In a combined chronic dietary toxicity/carcinogenicity study in rats, male and female Sprague Dawley rats were fed diets containing Chemical A at 0, 0.004, 0.06, 5 and 10 mg/kg/day for males and 0, 0.005, 0.07, 6 and 12 mg/kg/day for females for 98 weeks. No treatment-related changes were seen either in the absolute or relative weights of the testes, epididymides, prostate, uterus, ovaries, or the adrenal glands. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, uterus, ovaries, vagina, mammary, adrenal, thyroid or the pituitary glands. In a pre-natal developmental toxicity study, pregnant rats [Crl:COBS CD (SD) (BR)] received Chemical A at 0, 10, 20, and 100 mg/kg/day in 0.2% carboxy-methylcellulose by gavage on days 6 through 15 of gestation. Treatment-related findings seen only at the highest dose tested were increases in mean number of resorptions (1.8 vs 1.0 in the controls) and post-implantation loss (13.4% vs. 6.9% in the controls).  Treatment had no adverse effects on sex ratio or soft tissue abnormalities. No clinical signs related to cholinergic toxicity were noted.
In a developmental neurotoxicity study, Chemical A was administered to 27 female Crl:CD[(R)] (SD)BR IGS rats/treatment group in the diet at concentrations of 0, 0.30, 30 or 300 ppm from gestation day (GD) 6 through postnatal (lactation) day (PND) 21.  The average daily test article intake was 0, 0.026, 2.36, or 24.2 mg/kg/day during gestation and 0.039, 4.06, or 39 mg/kg/day from GD 6 through PND 21.  Preputial separation was delayed at the high dose (45.0 1.88 days) compared to controls (43.1 1.41 days).  Vaginal opening was also delayed by at the high dose (33.82.28 days) compared to controls (32.51.10 days).  The mean body weight at attainment of puberty was similar between the treated and control groups for males and females. For maternal systemic toxicity, the NOAEL was 24.2 mg/kg/day), the highest dose tested.  A LOAEL was not established. For maternal cholinesterase inhibition, the NOAEL was 0.026 mg/kg/day. The LOAEL was 2.36 mg/bw/day based decreases in plasma, RBC and brain cholinesterase activities. For offspring systemic toxicity, the NOAEL was 2.36 mg/kg/day. The LOAEL is 24.2 mg/kg/day based reduced body weight, reduced body weight gains and delayed sexual maturation in males and females. For offspring cholinesterase inhibition, the NOAEL was 0.026 mg/kg/day. The LOAEL was 2.36 mg/kg/day based on decreased plasma and RBC cholinesterase activities both sexes
In a two-generation reproduction study, male and female Sprague-Dawley rats were fed diets containing Chemical A at 0, 0.67, 6.69 and 35.15 mg/kg/day for males and 0, 0.77, 7.63 and 41.43 mg/kg/day for females in the diet for two successive generations. There were no treatment-related changes in testicular or ovarian weights. No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, coagulating glands, uterus, ovaries, vagina, cervix, or the pituitary glands. Slight decreases in male and female mating (86.7% vs. 96.7% in the control) as well as male and female fertility (73.1% vs. 89.7%) indices were seen at the highest dose in the second generation.  There was a trend towards prolonged gestation at the highest dose tested in both generations, i.e., 36% vs. 15% in the controls in first generation and 76% vs. 40% in the controls for the second generation.  None of these differences, however, reached statistical significance.  The sex ratio of the pups was slightly higher (p <0.05) for males at the highest dose tested (54.1%) compared to controls (52.7%) for the first generation.  No difference in sex ratio was seen for the second generation. Some adults in the high dose group from both the first and second parental generation exhibited tremors. Sexual maturation, estrous cycle length and periodicity, and sperm parameters were not evaluated in this study.   For parental/systemic toxicity, the NOAEL is 0.67 mg/kg bw/day and the LOAEL is 6.69 mg/kg bw/day based on decreased parental weight gain.  For offspring toxicity, the NOAEL is 0.67 mg/kg bw/day and the LOAEL is 6.69 mg/kg bw/day based on pup mortality and decreased weight gain.
Overall, the mammalian OSRI data do not support an interaction with the E, A or T pathways, with the exception of the slight delay in PPS seen in the rat developmental neurotoxicity study.

                 3.  Ecotoxicity OSRI Data
In an early life-cycle toxicity study with sheepshead minnow, Cyprinodon variegatus, based on the most sensitive endpoints (length, dry weight, wet weight), the NOEC and LOEC were 0.0043 and 0.008 mg/L a.i., respectively.

An early life-cycle toxicity study with Chemical A was conducted using fathead minnow. The nominal concentrations of  Chemical A selected for the definitive early life stage exposure were 0.094, 0.19, 0.38, 0.75, and 1.5 mg a.i./L. Statistically significant (p<0.05) decreases in larval growth were observed at all test concentrations. Egg hatching and larval survival were not affected by any test concentration of Chemical A.

The chronic effects of Chemical A on fathead minnows and brook trout (Salvelinus fontinalis) were determined in flow-through systems with constant toxicant concentrations. Fathead minnows exposed to the lowest concentration tested (0.0032 mg/L) from 5 days after hatch through spawning had a significantly higher incidence of scoliosis (i.e., sideways curvature of the spine) than the control (p= 0.05). Hatch of their progeny was reduced by 30 percent at this test concentration. Yearling brook trout exposed to 0.0048 mg/L and above began developing scoliosis and lordosis (i.e., inward curvature of the spine) within a few weeks. Growth of brook trout was substantially inhibited during the first 3 months at 0.0048 ug/L and above. Neurological symptoms were evident in brook trout at 0.0024 mg/L and above early in the tests, but were rarely observed after 4 or 5 months of exposure. Exposure of mature brook trout for 6 to 8 months to concentrations ranging from 0.0096 mg/L to the lowest tested (0.00055 mg/L) resulted in equally reduced growth rates for their progeny. Transfer of progeny between concentrations indicated that effects noted for progeny of both species at lower concentrations were the result of parental exposure alone and not the exposure of progeny following fertilization.

In a partial life-cycle toxicity test with fathead minnow with Chemical A, the NOAEC is 0.00092 mg/L and the LOAEC is 0.00195 mg/L based on a reduction in the number of eggs per spawn. Fish (59 days old at test initiation) were exposed for 116 days and reproduction was monitored with a 32-33 day post-hatch exposure of the F1 generation. There was no effect on F1 hatching success or 4-week growth (LOAEC >0.0078 mg/L), but survival of 4-week old larvae was affected at 0.0078 mg/L with a NOAEC of 0.0035mg/L.

In vivo acute toxicity testing with freshwater fish resulted in LC50 estimates as low as 0.090 mg/L; acute mortality data specific to fathead minnows indicate that this species is less sensitive (96-hr LC50=7.8 mg/L) than other freshwater species of fish. Acute toxicity testing with aquatic-phase amphibians (Rana boylii; LC50=7.5 mg/L) indicates that this taxon has a sensitivity similar to that of fathead minnow.  These data indicate that concentrations tested in the FSTRA and AMA may have overlapped levels capable of eliciting overt toxicity.
In a one-generation reproduction study with Northern bobwhite quail (Colinus virginianus) with Chemical A, the NOEC for the reproductive parameters was 32 mg/kg, the highest concentration tested. The NOEC for cholinesterase inhibition in blood serum was less than 8.3 mg/kg, the lowest concentration tested, for both males and females.

In a one-generation reproduction study with the mallard duck (Anas platyrhynchos), the NOAEC for reproductive parameters was 8.3 mg/kg diet, based on reduction in the number of eggs incubated as a percentage of eggs laid. The NOAEC for cholinesterase inhibition in blood serum was less than 4.02 mg/kg, the lowest concentration tested, for both males and females. Additionally, brain cholinesterase levels for females were depressed compared to the controls at all treatment levels.

In a chronic life-cycle toxicity test with the freshwater invertebrate waterflea (Daphnia magna) with Chemical A, there were no effects on growth and reproduction at Chemical A concentrations of <=0.00017 mg/L. There was 100% mortality at test concentrations, >=0.00032 mg/L. The study author reported NOAEC was 0.00017 mg/L.

In a chronic toxicity test with an estuarine/marine invertebrate, i.e., mysid shrimp (Americamysis bahia) with Chemical A, the most sensitive endpoint was growth (dry weight) with a NOAEC and LOAEC value of 0.0.00023 and 0.00042 mg a.i./L, respectively. There was no effect on reproduction or survival up to 0.0016 mg a.i./L.

No additional data with frog or other amphibian species have been submitted.
Overall, the only effect observed in the OSRI ecotoxicity data that could be attributed to an interaction with the E, A or T pathways was a reduction in the number of eggs in the freshwater fish and mallard duck.  Egg reduction, however, is not an endocrine-specific endpoint and may result from overt toxicity.

Discussion
     i. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis
EDSP Tier 1 screening data and OSRI were evaluated together to determine whether Chemical A 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 17 below summarizes the results of the available EDSP Tier 1 screening assay result and OSRI results for determining the potential for Chemical A 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.

Table 17:  Estrogenic/Anti-Estrogenic Pathway for Chemical A
Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chemical A [a]
Study Type /
Literature Citation
ER Binding
ER Activation
Steroidogenesis
Uterine Weight
Ovarian Weight
Ovarian/Gonad Staging and Histopathology
Pituitary Weight
Estrous Cyclicity[b]
Age & Weight at VO
Fertility/Fecundity
Vitellogenin
Overt Toxicity Observed[C]
                                  EDSP Tier 1
ER Binding

Equivocal.

ERTA

No effect

Aromatase

Equivocal

Steroidogenesis

No effect

Uterotrophic

No effect

Female Pubertal Rat

No effect
No effect
No effect
No effect
No effect
No effect
No effect

FSTRA

H: follicle atrophy

H: 80%
♀L: 63%
♀H: 71%
X
                                     OSRI
Kijoma et al. ER reporter assay

Positive

Subchronic toxicity (Rat)

No effect
No effect
No effect
No effect

Chronic toxicity (Rat)

No effect
No effect
No effect

Chronic toxicity (Dog)

No effect
No effect
No effect

Two-generation reproduction  (Rat)

No effect
No effect

H:↓37-59%

X
Developmental Neurotoxicity (Rat)

 1.3 days (p<0.05)

X
a Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatment.  Arrows (↓ or ↑) indicate the direction of the response.  A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this 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 potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity

The EDSP Tier 1 battery of assays measure endpoints that are considered to be complementary within an assay and/or redundant across assays. The results for ER binding and aromatase inhibition were considered equivocal and were potentially confounded by the solubility limit of Chemical A. In a published literature study, submitted as OSRI (Kojima, 2005), Chemical A was weakly estrogenic (an EC10 of 4.6 x 10[-4] M) when the ER transactivation potential was evaluated in the E-CALUX test system; however, the chemical did not transactivate the ER in the EDSP ERTA assay when tested up to a cytotoxic concentration. In the steroidogenesis assay, Chemical A did not alter estradiol production. The equivocal in vitro responses in the ER binding and aromatase assays were not corroborated by altered organ or tissue level responses in the mammalian in vivo assays that would otherwise have been complementary. Chemical A did not induce a response in the uterotrophic assay and no estrogen-related endpoints were altered in the female pubertal assay.  In the FSTRA, effects on vitellogenin, gonadal histopathology, fertility, fecundity, and bodyweight were observed at concentrations where there was clear evidence of overt toxicity that included abnormal behavior (abnormal swimming) and significant body weight reductions which may be consistent with cholinergic toxicity. Given the directionality (decreases in the measured endpoints) of the FSTRA responses, the effects may not necessarily be reflective of an endocrine-mediated response, but rather may reflect a compromised organism with limited ability to maintain reproductive function and homeostasis.  Although control performance in this study was not entirely consistent with performance criteria, for fecundity and fertility, the study does not provide consistent evidence of estrogen pathway interaction across the various levels of biological organization measured in this study.
OSRI in the form of Part 158 mammalian toxicity studies do not indicate that Chemical A alters structural responses at the organ or tissue level.  No organ weight or histopathological effects were seen in uterus or ovary in subchronic (rat) or chronic (rat and dog) toxicity studies.    In a multi-generation study in rats, the only potential estrogen pathway-related effect observed was a decrease in fertility, which is a non-specific effect and may not be an endocrine response.  In a rat developmental neurotoxicity study, a very slight delay in vaginal opening (1.3 days) was observed in the offspring.  In the OSRI ecotoxicity data, a reduction in the number of eggs was observed in freshwater fish and mallard duck, however, this is not endocrine-specific endpoint and the direction of the response (egg reduction) may be reflective of overt toxicity.  An integration of data from the EDSP Tier 1 and OSRI studies do not indicate that Chemical A interacts with the E pathway. Chemical A was not an ER agonist up to cytotoxic concentrations in the ERTA assay.  The equivocal responses in the ER binding and aromatase assays occurred at concentrations near the limit of solubility for the test compound and did not translate into effects in higher levels of biological organization in the mammalian Tier 1 in vivo studies.  A slight delay in vaginal opening was observed in a developmental neurotoxicity study in rats, but the delay was only 1.3 days and considered inconsequential.  Additionally, no E pathway-related effects were observed in the FSTRA in the absence of overt toxicity.   Based upon WoE evaluation of EDSP Tier 1 data and OSRI, the absence of robust complementarity and redundancy in responses do not support potential interactions with estrogen signaling pathway.

Effects on Androgen Pathway: Table 18 below summarizes the results of the available EDSP Tier 1 screening assay results and OSRI for determining the potential for Chemical A 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 A demonstrated the potential to interact with the AR and was classified as a binder in the AR binding assay.  Chemical A slightly inhibited testosterone production in H295R cells in the steroidogenesis assay.  In the in vivo Hershberger assay, Chemical A showed a dose-dependent positive anti-androgenic response in sex accessory organ weights in castrated rats at all three doses evaluated (15, 47 and 150 mg/kg/day).  However, when Chemical A was tested in the male pubertal assay using animals with an intact HPG axis, no androgen-related effects were observed at doses up to 100 mg/kg/day.  A number of endocrine responses were observed (e.g., decrease in female VTG, fecundity, GSI, male tubercles) at the highest concentration in the FSTRA, there was also pronounced overt toxicity that included abnormal behavior and significant body weight reductions consistent with cholinergic intoxication.   Given the directionality of the FSTRA responses (i.e., decreases in the measured endpoints), the effects found at the high concentration in the FSTRA may not necessarily be reflective of an endocrine-mediated response, but rather a reflection of a compromised organism with limited ability to maintain reproductive function and homeostasis.  Although in male fish, overt toxicity was not observed at the intermediate concentration, possible endocrine responses were limited to two effects that lacked diagnostic specificity (i.e., altered GSI and histology).
In the Part 158 studies submitted as OSRI, no effects on organ weight or histopathology of the testes, epididymis or sex accessory organs were observed with subchronic or chronic exposure of Chemical A in the rat or in the chronic toxicity study in dogs.   No androgen pathway-related effects were observed in the 2-generation rat reproduction study.  In a rat developmental neurotoxicity study in rats, a slight delay (less than 2 days) in preputial separation was observed.
There is some evidence of an interaction when evaluating the androgen pathway responses across all the considered studies.  Chemical A interacts with the AR in the binding assay and decreases testosterone production in the steroidogenesis assay.  In the Hershberger assay, Chemical A acts as an androgen antagonist causing dose-related decreases in androgen-responsive tissue weights. No effects were found in the Tier1 male pubertal assay. However, a slight delay in preputial separation was also observed in a developmental neurotoxicity study (DNT) in rats.  Although these responses are consistent with the potential interaction with the A signaling pathway (via androgen antagonism and perturbation of steroidogenesis), there is some uncertainty around the observed responses.  The in vitro effects were seen only at a high test concentration that was near the limit of solubility for the compound.  In the FSTRA, there were only two endpoints affected in the absence of overt toxicity, and these are nonspecific responses and could be due to endocrine or non-endocrine mechanisms. Effects in the Hershberger assay were not observed in the male pubertal assay (rats with intact HPG) which was conducted at similar doses.
The in vitro responses in the AR binding and steroidogenesis assays (albeit at high concentrations) and the tissue responses in the Hershberger indicated that Chemical is potentially interacting with the A pathway.  In organisms with an intact HPG axis, Chemical A showed no androgen related effects in the male pubertal assay, and in FSTRA, several endpoints were altered that could be consistent with androgen antagonism; however, many were in the presence of overt toxicity.
For the A signaling pathway, the in vitro assay results suggest that Chemical A has the potential to interact with the AR and is able to alter steroidogenesis resulting in a decrease in testosterone production.  Tissue level responses (decreased organ weights) consistent with AR binding and/or decreased testosterone production were observed in the Hershberger assay.  However in the intact in vivo model (e.g. pubertal male), presumably with compensatory mechanisms, no androgen related effects were observed.  In the FSTRA, several endpoints were altered that could be consistent with androgen antagonism; however, many were in the presence of overt toxicity.   The Part 158 toxicology data did not provide convincing evidence of an in vivo interaction with the A pathway.

Table 18:  Androgenic/Anti-Androgenic Pathway for Chemical A
Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chemical A[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
Overt Toxicity Observed[b]
                                  EDSP Tier 1
AR Binding
Binder

Steroidogenesis

 Test.

Hershberger

L:   SV 29%
VP 26%

M: SV 37%
VP  43%
 LABC  21%

H:  SV 86%
VP  77%
LABC 45%
CG  69%
GP  20%

Male Pubertal Rat

No
effect
No
effect
No
effect
No
effect
No
effect
No
effect
No
 effect
No
effect

FSTRA

M: 29%
H: 31%
L,M,H:  spermatogonia

H: 74% nuptial tubercle score

No effect
X

                                     OSRI
Subchronic toxicity (Rat)

                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect

Chronic toxicity (Rat)

                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect

Chronic toxicity (Dog)

                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect
                                      No
                                    effect

                                      No
                                    effect

Two-generation reproduction  (Rat)

                                      No
                                    effect
                                      No
                                    effect

                                      No
                                    effect

                                      No
                                    effect

Developmental Neurotoxicity (Rat)

                                   H: 1.9 d

                                       X
a Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatment. Arrows (↓ or ↑) indicate the direction of the response.  A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.   SV= seminal vesicle; VP= ventral prostate; LABC= levator ani-bulbocavernosus muscle; CG= Cowper's gland; GP= glans penis
b  An "X" in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity.

         ii. 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 19 summarizes the results of the available EDSP Tier 1 studies and endpoints relevant for determining the potential for Chemical A 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 19 also includes HPT-relevant findings from data evaluated as OSRI.
	In the EDSP Tier 1 battery results, the only observed response that could be attributed to a potential interaction with the HPT axis was a slight decrease (↓23% and 17%) in serum T4 levels observed in female pubertal rats treated with Chemical A at 50 and 100 mg/kg bw/day, respectively. A small decrease (↓15%) in T4 was also seen in the male pubertal assay at 50 mg/kg bw/day, but not at 100 mg/kg bw/day and was not considered biologically significant.  The depression in T4 was not associated with complementary alterations in TSH levels or changes in thyroid weights, pituitary weights, or thyroid histopathology in the male or female pubertal assays that might be anticipated if the compound were interacting with the HPT axis. Furthermore, the exposure period in the male pubertal assay is 10 days longer than the female pubertal assay, yet no compelling thyroid effects were observed in the male.   Thyroid gland hypertrophy was observed in the AMA at the highest treatment level (0.820 mg a.i./L); however, no other noteworthy effects were reported.  Based on these data, there was not compelling evidence for potential thyroid interaction in the AMA.  There were also no changes in thyroid weights, pituitary weights or thyroid histopathology in the mammalian Part 158 studies evaluated.  These studies included a subchronic study in rats treated with Chemical A for 13 weeks at doses up to 168 mg/kg bw/day and 2-year rat study and 1-year dog study treated with doses up to 10 and 9 mg/kg bw/day, respectively.

Table 19:  Thyroid Pathway for Chemical A
Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chemical A[a]
Study Type /
Literature Citation
Hormones
(T4 and TSH)
Pituitary Weight
Thyroid Weight
Thyroid  Gross and Histopathology
Frog Development Stage
Hind Limb Length
Snout to Vent Length
Overt Toxicity Observed[b]
                                  EDSP Tier 1
Male Pubertal (Rat)
L: ↓T4 23%
H: ↓T4 17%
No Effect
No Effect
No Effect

Female Pubertal (Rat)
L:  ↓T4 15%
No Effect
No Effect
No Effect

AMA (Frog)

Equivocal
No Effect
No Effect
No Effect

                                     OSRI
Two-generation reproduction  (Rat)

No Effect
No Effect
No Effect

Subchronic toxicity (Rat)

No Effect
No Effect
No Effect

Chronic toxicity (Rat)

No Effect
No Effect

Chronic toxicity (Dog)

No Effect
No Effect

a Key to responses:  Key to responses: [L] Low treatment, [M] Medium treatment, [H] High treatment.  Arrows (↓ or ↑) indicate the direction of the response.  A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.
b  An "X" in this column indicates that the potential endocrine effect(s) observed in the assay occurred in the presence of overt toxicity.
Evidence of slight T4 depression in the female pubertal rats was not corroborated by complementary changes in TSH, thyroid weight or thyroid histopathology.  The decrease in T4 was not consistent with the magnitude of effects typically seen with thyroid toxicants.  There were also no biologically relevant changes in T4 levels in the pubertal males which are treated for 10 days longer than female rats in the pubertal assay.  The lack of thyroid pathway-related effects in the AMA and mammalian Part 158 studies reinforce the absence of thyroid interaction.  Based upon WoE evaluation of EDSP Tier 1 data and OSRI, Chemical A does not demonstrate a potential to interact with the thyroid pathway.

Conclusions for Chemical A
All eleven assays comprising the EDSP Tier 1 battery were available for Chemical A. Chemical A 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 A 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 and whether overt toxicity was present were considered. This is particularly important in the case of Chemical A, given that it belongs to the organophosphate class of chemicals that results in cholinergic toxicity.

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) did not reveal convincing evidence of an interaction with the E or T pathways.  There was reasonable complementarity within assays and redundancy among responses across assays in terms of a lack of clear and consistent responses for E across different levels of biological organization. For the T pathway, the AMA was negative and only small changes in T4 were seen in the pubertal assays at doses that would be expected to cause significant cholinesterase inhibition. Although there were responses in FSTRA that could be indicative of an interaction with the estrogen signaling pathway, they were only found in the presence of overt toxicity.
With respect to the A signaling pathway, the in vitro responses in the AR binding and steroidogenesis assays (albeit at high concentrations) and the tissue responses in the Hershberger indicated that Chemical is potentially interacting with the A pathway.  In organisms with an intact HPG axis, Chemical A showed no androgen related effects in the male pubertal assay, and in FSTRA, several endpoints were altered that could be consistent with androgen antagonism; however, many were in the presence of overt toxicity.   The Part 158 toxicology data did not provide convincing evidence of an in vivo interaction with the A pathway.

Case Study  --  Chemical S

A. Introduction
Chemical S is an organometal compound and has a mode of action involving inhibition of oxidative phosphorylation at the site of dinitrophenol uncoupling and as such it affects the production of energy in the form of adenosine triphosphate, ATP.  In the case of Chemical S, the issues included: potential interactions with more than one endocrine modality; potential endocrine-related effects in the presence of overt toxicity; challenges encountered in dose selection (overt toxicity); and endpoint sensitivity (e.g., endocrine vs. overt toxicity).
The available information considered to determine the potential interaction of Chemical S 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.

B. Data Available for Chemical S
Chemical S is a relatively insoluble compound with a high log octanol-water partition coefficient (log Kow>5) and organic carbon partition coefficient (Koc~321,000 L/kg).  The compound is relatively stable to abiotic routes of degradation (i.e., hydrolysis and photolysis) and to aerobic metabolism; however, Chemical S can degrade slowly via anaerobic metabolism.  Therefore, Chemical S is expected to persist in the environment and given its high Koc is likely to partition to sediments.  Although the compound has a relatively high Kow, fish do not demonstrate appreciable bioconcentration factors suggesting that the compound may be metabolized by fish and/or its uptake may be limited.  The low solubility in water combined with the high Kow and Koc values render this chemical difficult to test in aquatic systems as the chemical would readily sorb to test containers.
C. Tier 1 Screening Assays for Chemical S
The Tier 1 studies submitted in response the test order for Chemical S are shown below in Table 20 and the relevant study details and findings are summarized below.
Table 20:  Summary of Tier 1 Screening Assays for Chemical S
                                 Tier 1 Assay
                                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
                                  Negative[2]
Steroidogenesis Assay (Human cell line H295R)
OSCPP 890.1550;
OECD 456
                                      No
                                   Negative
Aromatase Assay (human recombinant microsomes)
OSCPP 890.1200
                                      No
                                   Equivocal
Uterotrophic Assay (Rat)
OSCPP 890.1600;
OECD 440
                                      No
                                   Negative
Hershberger Assay (Rat)
OSCPP 890.1400;
OECD 441
                                      No
                                Negative for A
                             Positive for Anti A
Pubertal Female Assay (Rat)
OSCPP 890.1450
                                      No
                            Positive for E pathway
                            Negative for T pathway
Pubertal Male Assay (Rat)
OSCPP 890.1500
                                      No
                            Negative for A pathway
                            Negative for T pathway
Fish Short-term Reproduction Assay
OSCPP 890.1350;
OECD 229
                                      No
                          Possible for A pathway[3]
Amphibian Metamorphosis Assay (Frog)
OSCPP 890.1100;
OECD 231
                                      No
                                 Inadequate[4]
[1]Classified as "Not Interactive" according to the Test Guideline OSCPP 890.1250
[2] Classified as "Non-Binder" according to the Test Guideline OSCPP 890.1150
[3] Effects coincide with decreases in survival which confounds interpretation
[4] Indequate exposure
              i.         ER Binding Assay (Rat uterine cytosol)
In the ER binding assay (98.75% a.i.), uterine cytosol from Sprague Dawley rats was used as the source of ER for a Competitive Binding Experiment, which was conducted to measure the binding of a single concentration of tritiated [[3]H]-17β-estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10[−12] to 10[−5] M) of Chemical S.  A solubility test showed that Chemical S was not soluble in dimethyl sulfoxide (DSMO) or ethanol at concentrations >10[-5] M.  Ethanol was used as the solvent vehicle at a final assay concentration of <3% (v/v).  A total of three valid runs were performed, and each run included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17-β-estradiol as the natural ligand reference material.
A saturation binding experiment demonstrated that the ER was present in the rat uterine cytosol at a reasonable concentration and functioning with appropriate affinity for the 17β-estradiol ligand.  The dissociation constant (Kd) for [[3]H]-17β-estradiol was 0.1153 nM, and the estimated average maximum binding (Bmax) was 45.75 fmol/100 μg protein for the single run with the batch of uterine cytosol that was prepared.  The Kd and the Bmax were within the expected ranges.  Confidence in these numbers is high based on visual inspection of the goodness of fit.  The specific binding reached a plateau, and the maximum non-specific binding was less than 24% of total binding.
In a competitive binding experiment, the estimated average specific binding affinity (reported as Log IC50) was −8.94 Log M for 17β-estradiol and −6.11 Log M for the weak positive control (19-norethindrone).  Compared to 17β-estradiol, the mean relative binding affinity (RBA) for the weak positive control was 0.150%.
All of the performance criteria for 17β-estradiol were met across the three runs of the competitive binding assay.  The guideline does not provide performance criteria for 19-norethindrone, the weak positive control.  The non-specific binding was approximately <=0.5%, and the curve for the reference material showed that increasing concentrations of unlabeled 17β-estradiol generally displaced [[3]H]- 17β-estradiol in a manner consistent with one-site binding, as indicated by a mean Hill slope of approximately −0.9 to −0.8.

Based on the results of three runs, Chemical S is classified as Not Interactive in the ER binding assay.

              ii.  ii.   ER Transcriptional Activation Assay
In the ER transcriptional activation assay, hERα-HeLa-9903 cells  were exposed to Chemical S (97.82% a.i.) at logarithmically increasing concentrations from 10[−13] to 10[−6] M in 0.1% ethanol for approximately 24 hours in two independent runs.  The experiments were performed using 96-well plates and each concentration was tested in 6 wells/plate per run.

A preliminary cytotoxicity test showed that Chemical S was cytotoxic in this assays system at concentrations of 10-6 M (35% viability) and above.  The highest concentration tested in the ERTA assay was 10[-6] M.  Cells were exposed to the test agent for 24+-2 hours at 37+-1°C to induce reporter (luciferase) gene products.  Luciferase expression in response to activation of the estrogen receptor was measured using a luciferase assay.
The performance criteria were met for the reference chemicals 17β-estradiol, 17α-estradiol and corticosterone,   however, the very weak agonist, 17α-methyltestosterone, did not achieve 50% transcriptional activation, therefore a PC50 could not be calculated.  Additionally, the response of the cells to 17α-methyltestosterone was weaker than the expected response, and the Log PC10 for the two runs (-5.7 and -5.8) fell below the performance criteria range (-8.0 to -6.2) for this reference chemical.  Chemical S was cytotoxic at 10[-6] M in both runs (29 - 46.5% viability).    The highest mean induction value as expressed as the percentage of response induced by the positive control (RPCmax) for Chemical S in both runs was 2.9%, which is less than the 10% response required for a positive response.   There was no evidence of ER transcriptional activation by Chemical S up to 10[-7] M.
Chemical S was negative for ER transcriptional activation in this test system.

              iii. iii. AR Binding Assay (Rat prostate cytosol)
In the AR binding assay, ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment.  The competitive binding assay was conducted to measure the binding of a single concentration of [[3]H]-R1881 (1 nM) in the presence of increasing concentrations (10[−11] to 3.33 x 10[−5] M) of Chemical S (97.85%).  Ethanol was used as the solvent vehicle for Chemical S, and the reference chemicals (dexamethasone and R1881), at a final assay concentration of 4.19% (v/v).  A total of three runs were performed.  Each run included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.
The saturation binding  experiment demonstrated that the AR was present in the rat ventral prostate cytosol at a reasonable concentration and functioning with appropriate affinity for the R1881 ligand.  The dissociation constant (Kd) for [[3]H]-R1881 was 1.255 nM, and the estimated Bmax was 15.75 fmol/100 μg protein for the single run with the batch of prostate cytosol that was prepared.  The Kd and the Bmax were within the range for the performance criteria.  The specific binding reached a plateau, and the maximum non-specific binding was less than 18% of total binding.  The Scatchard plots for the saturation binding assay data were approximately linear.
In the competitive binding experiment, the estimated average Log IC50 was −8.79 for R1881 and −4.34 for the weak positive control (dexamethasone).  Compared to R1881, the mean relative binding affinity (RBA) for the weak positive control was 0.0036%.  No precipitation of the test compound was visually observed at <=3.33 x10[−5] M.
All of the performance criteria for R1881 and dexamethasone were met across the three runs.  The non-specific binding was approximately <=2%, and the curve for the reference material showed that increasing concentrations of unlabeled R1881 generally displaced [[3]H]-R1881 in a manner consistent with one-site binding, as indicated by a mean Hill slope of approximately −1.0.  The lowest portion of the Chemical S across all runs was >75% at 3.3 x 10[−5] M (highest concentration tested).

Based on the results of three runs, Chemical S is classified as a "Non-binder" in the AR binding assay.

              iv. iv. Steroidogenesis Assay (Human cell line H295R)
In a steroidogenesis assay, H295R cells cultured in vitro in 24-well plates were incubated with Chemical S (97.82% purity) at log concentrations from 0.00001 to 1 uM in triplicate for 48 hours.  The solvent vehicle for the test chemical was ethanol, at a final assay concentration of 0.075% (v/v).  Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards.  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. Positive controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.
A total of 12 independent runs of the steroidogenesis assay were reportedly conducted; however, only the results from 5 of the test runs were reported.  The remaining seven tests reportedly failed because either the QC plates did not meet the guideline criteria or because there was excessive variability in the test plates.  Increased cytotoxicity occurred in the Chemical S-treated cells at 1 uM (1.9-21.0% viability) in all five runs, therefore, data from the 1 μM concentration were not evaluated.  Precipitation was also noted at this concentration in Run 1.  No other cytotoxicity was noted.
The performance criteria were generally met for the test plates and for the QC plates, with the following minor exceptions.  Estradiol production in the blank and solvent control (SC) wells was less than 40 pg/mL in Runs 4 (36-37 pg/mL) and 5 (33-34 pg/mL), but the basal estradiol production was >=2.5-fold above the minimum detection limit (MDL) of the analytical method (10 pg/mL).  Inhibition of estradiol by 1 uM prochloraz slightly exceeded the 0.5-fold performance criteria in four runs (0.51- to 0.58-fold), and induction of testosterone by 10 uM forskolin was below the 2-fold the performance criteria in all five runs (1.32- to 1.68 fold).  However, when the background hormone concentrations were subtracted, 10 uM forskolin induced testosterone an average of 2.56-fold.  In addition, the between-plate coefficient of variation (CV) for testosterone was 38.2%, which is above the recommended limit of 30%.
There were slight decreases (p<=0.05) in testosterone levels (0.92- to 0.93-fold) at Chemical S concentrations of 0.0001-0.1 uM in Run 5, but the effect was not dose-dependent and significant decreases in testosterone were not noted in the other four runs at these concentrations.  The only other significant changes (p<0.05) in testosterone were a slight decrease (0.86-fold) in Run 1 at and a slight increase (1.18-fold) in Run 2, both at 0.00001 uM.  For all five runs, the average fold difference in testosterone concentrations was 0.96- to 1.02-fold at Chemical S concentrations of 0.00001-0.1 μM.  When hormone levels were normalized for cell viability, there were no significant effects on testosterone levels at any Chemical S concentration in any run.
For estradiol, the only significant change compared to the SC was a 0.87-fold reduction at the 0.1 μM concentration in the third run.  The average fold difference in estradiol concentrations was 1.00- to 1.05-fold at Chemical S concentrations of 0.00001-0.1 μM.  When estradiol levels were normalized for cell viability, there was a minor increase (p<0.05) in estradiol of 1.08-fold in Run 1 at 0.0001 uM, and a slight decrease (p<0.05) of 0.87-fold in Run 3 at 0.1 uM.
Based on the hormone responses in three runs, Chemical S was negative in the steroidogenesis assay.

              v.      Aromatase Assay (Human Recombinant Microsomes)
In an in vitro aromatase (CYP19) assay, Chemical S (97.85% a.i.) was incubated with human recombinant aromatase and tritiated androstenedione ([[3]H]-ASDN) in 1% ethanol at log concentrations from 10[−10] to 10[−5] M for 15 minutes to assess the effect of Chemical S on aromatase activity.  The 10[−5] M concentration of Chemical S was the highest test concentration due to solubility issues at higher concentrations.
Aromatase activity was determined by measuring the amount of tritiated water produced at the end of a15-minute incubation for each concentration of test chemical.  Tritiated water was quantified using liquid scintillation counting (LSC).  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) using a known inhibitor (i.e., 4-OH ASDN), and the Chemical S Chemical Series (10[−10] to 10[−5] M) with three replicates per concentration.
Aromatase activity in the full activity controls ranged from0.309 to 0.455 nmol∙mg-protein[−1]∙min[−1] for the three test runs, with a mean +- standard deviation of 0.388 +- 0.048 nmol∙mg-protein[−1]∙min[−1].  Activity in the background controls averaged 0.0028 +- 0.001 nmol∙mgprotein[−1]∙min[−1], or 0.71% of the full control activity.  The response of the full activity controls and background controls were acceptable for each run.
For the positive control substance (4-OH ASDN), aromatase activity averaged 0.387 +- 0.018 nmol∙mg-protein[−1]∙min[−1] at the lowest tested concentration 10[−10] M and 0.0026 +- 0.0003 nmol∙mg-protein[−1]∙min[−1] at the highest tested concentration 10[−5] M.  The estimated Log IC50 for 4-OH ASDN averaged −7.23 Log M and the Hill Slope was −0.996.
For Chemical S, aromatase activity averaged 0.407 +- 0.061 nmol∙mg-protein[−1]∙min[−1] at the lowest tested concentration 10[−10] M and 0.270 +- 0.022 nmol∙mg-protein[−1]∙min[−1] at the highest tested concentration 10[−5] M.  The Log IC50 could not be calculated, as 50% inhibition was not achieved with Chemical S.  However, with the exception of response seen at the 10[−9] M (88.5% activity) , the limited inhibition observed for Chemical S was generally dose responsive, declining from 101.7% full control activity at  10[−7.5] M down to 68.8% activity at 10[−5.5] M.  The average lowest portion of the curves across the three runs was between 50 and 75% activity, indicating an equivocal response in this assay (Figure 12 below).
Figure 12.  Inhibition Response Curves for Chemical S From Each Test Run.

Based on the results of three runs, Chemical S is classified as Equivocal in the aromatase assay.

  	  	vi.  Uterotrophic Assay (Rat)
In the in vivo Uterotrophic Assay conducted to screen for potential estrogenic activity, Chemical S (97.85% purity) in corn oil was administered daily via oral gavage to groups of six adult ovariectomized, female Wistar (RccGab:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg bw/day for three days from post-natal days (PND) 61/62 to 63/64.  A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) at 0.3 ug/kg bw/day by subcutaneous injection.  Body weights were determined daily.  All animals were euthanized and necropsied on PND 64/65 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.
All animals survived until scheduled termination.  Clinical signs of toxicity were limited to salivation in 1/6 and 4/6 rats at 10 and 75 mg/kg bw/day, respectively.  Terminal body weight was decreased (not significant, NS) by 5% in the 75 mg/kg bw/day group compared to vehicle controls.  This group displayed an overall (Days 1-4) body weight loss (p<0.05) of -0.68 g compared to the 10.37 g weight gain by the controls.  Body weights in the 10 mg/kg bw/day group were comparable to the control throughout the study; however, overall body weight gain was decreased (NS) by 42% compared to controls.  Uterine weights (wet and blotted) in the Chemical S treatment groups were comparable to the vehicle controls. Body weights and overall body weight gain in the positive control (EE) group were comparable to the controls.  As expected, the absolute wet and blotted uterus weights for the EE group were increased (p<0.01) by 246 and 200%, respectively, over the 2 days of necropsy.  The high-dose level was an adequate dose based on overall body weight loss during the treatment period.
No statistically significant increase in uterine weight was seen in this study. Hence, Chemical S was negative in the Uterotrophic assay.

              vi. vii.  Hershberger Assay (Rat)

The dose levels for the in vivo Hershberger assay were selected based on the results from a range-finding study in Wistar rats (5/sex/dose).  Chemical S was administered via oral gavage at doses of 0, 10, 50, or 100 mg/kg bw/day for 28 days.  At 100 mg/kg bw/day, one female was found dead on Day 19.  All other animals survived to scheduled sacrifice.  Salivation was observed in 2 males at 10 mg/kg bw/day and in all animals at 50 and 100 mg/kg bw/day.  Additionally, one female at 100 mg/kg bw/day displayed dyspnea and gasping.  Body weights, body weight gains, and food consumption were decreased in the 100 mg/kg bw/day males for 2-4 weeks of the 4 week treatment period.  Although there were no significant decreases in body weights observed in the females at any dose compared to controls, body weight gains in the 100 mg/kg bw/day male group were decreased during Week 1-2.  At 100 mg/kg bw/day, possible target organ effects based on organ weight changes were noted in the testes, levator ani-bulbocavernosus  (LABC) and Cowper's gland, liver, and spleen. Certain biochemical disturbances such as increases in leukocyte counts, total bilirubin, ALT activity, and phosphorous levels were noted at 50 and 100 mg/kg/day; these effects by sex were not specified in the study report.  Based on these results, a high dose of 75 mg/kg bw/day was selected for the Hershberger Assay.
In a Hershberger Assay screening for androgenic activity, Chemical S (97.85% purity) in corn oil was administered daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan:WIST) rats at dose levels of 0 (vehicle), 10, or 75 mg/kg/day for 10 days.  The test included a vehicle control group and an androgenic positive control group consisting of six castrated rats exposed to 0.4 mg/kg bw/day of testosterone propionate (TP) by subcutaneous (s.c.) injection.
In a second phase of the Hershberger Assay screening for potential anti-androgenic activity, Chemical S (97.85% purity) in corn oil was administered daily via oral gavage to groups of six 51-day old, castrated male Wistar (RccHan: WIST) rats dose levels of 10, 40, or 75 mg/kg bw/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg bw/day by s.c. injection.  The negative control group for anti-androgenic activity consisted of six castrated rats dosed with TP at 0.4 mg/kg bw/day by s.c. injection, and the antiandrogenic positive control group consisted of six castrated rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg bw/day in conjunction with a s.c. dose of TP (0.4 mg/kg bw/day).
For both assays, body weights were determined daily and overall (Days 1-11) food consumption (g/rat/day) was determined.  Animals were dosed for 10 consecutive days and euthanized approximately 24 hours after the final dose administration to determine weights of the five androgen-dependent accessory sex tissues (i.e., seminal vesicles, ventral prostate, levator ani-bulbocavernosus (LABC), Cowper's glands or glans penis), liver, kidney, and adrenal weights.  All animals in both studies survived until scheduled termination.
Clinical signs of toxicity in both phases of the assay were limited to salivation in 3/6 and 6/6 rats in the 10 and 75 mg/kg bw/day groups, respectively, in the androgen agonist study, and in 4/6 and 6/6 rats in the 40 and 75 mg/kg bw/day groups in the anti-androgen study.  There were no treatment-related effects on weights of liver, kidneys, adrenals or any the accessory sex tissues of Chemical S-treated animals.  In the androgen agonist assay, there were no statistically significant (NS) differences in body weights at either dose of Chemical S, although overall body weight gain (Days 1-11) was decreased (p<0.05) by 17%, and overall food consumption was decreased (p<0.01) by 6% at 75 mg/kg bw/day.  Although seminal vesicle weights were increased (p<0.05) by 21% at 10 mg/kg bw/day, this solitary finding occurred only at the lowest dose.  As expected, the TP positive control group had increases (p<0.01) in the weights of the seminal vesicles (↑1097%); ventral prostate (↑787%); LABC (↑114%); Cowper's glands (↑551%); and glans penis (↑76%), indicating that the test system was sensitive to an androgenic response.  Because significant weight increases were not observed in two or more androgen-dependent tissues, Chemical S is not considered to be an androgen agonist at <=75 mg/kg/day.
In the anti-androgen assay, there were decreases (p<0.05) of 5-7% in body weights on Days 711% at 40 mg/kg bw/day+TP and 7-10% on Days 4-11 at 75 mg/kg bw/day+TP.  Overall body weight gains were also decreased (p<0.01) by 29 and 45% at 40 and 75 mg/kg bw/day, respectively, and food consumption was decreased (p<0.01) by 11-33% in all Chemical S-treated groups.  There were no treatment-related effects on weights of liver, kidneys or adrenals.  However, significant weight decreases (p<0.05 or <0.01) were noted in the ventral prostate (↓19%) and glans penis (↓16%) at 10 mg/kg bw/day.  At 40 mg/kg bw/day+TP, tissues weights were decreased (p<0.01) by 34% in the seminal vesicles, 40% in the ventral prostate, 16% for the LABC, 27% for the Cowper's glands, and 20% for the glans penis.  At 75 mg/kg bw/day+TP, tissues weights were decreased (p<0.01) by 23% in the seminal vesicles, 24% in the ventral prostate, and 23% for the LABC.  These decreases were not generally dose dependent with the exception of the ventral prostate and the glans penis.
As expected, the positive FT+TP control group had decreases (p<0.01) in weights of the seminal vesicles (↓81%), ventral prostate (↓72%), LABC (↓47%), Cowper's glands (↓67%), and glans penis (↓35%) compared to the negative TP control group.  Significant decreases in the weight of two or more androgen-dependent tissues occurred at doses from 10 to 75 mg/kg bw/day; however, there was overt toxicity at the 75 mg/kg/day dose level. Chemical S is considered to be a potential anti-androgen at the dose level of 10 mg/kg bw/day (absence of overt toxicity) under the conditions of this assay.
The Guideline criteria for %CV in organ weights were met in all cases for the vehicle/negative control and high dose groups.
Statistically significant increases in organ weights were not seen in two or more of the five androgen responsive tissues in the androgen agonist assay.  Chemical S was negative for androgenic activity.
Statistically significant decreases in ventral prostate and glans penis weights were observed at 10 mg/kg/day in the androgen antagonist assay in the absence of overt toxicity. Chemical S is considered positive for potential anti-androgenic activity; data are summarized in Tables 21 through 25.
 Chemical S was negative for androgenic activity, and positive for potential anti-androgenic activity at doses not associated with overt toxicity in the Hershberger assay.
Table 21:  Chemical S - Selected Group Mean (+-SD) Body Weights and Cumulative Body Weight Gains (g) in the Androgen Agonist Assay [a]
                                  Study Day #
                                     Dose

                                Vehicle Control
                               Positive Control
                              TP (0.4 mg/kg/day)
                                  Chemical S
                                (10 mg/kg/day)
                                  Chemical S
                                (75 mg/kg/day)

                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       1
                                       6
                                     225.9
                                     10.2
                                       6
                                     226.6
                                      8.2
                                       6
                                     227.5
                                      8.0
                                       6
                                     224.8
                                      7.2
                                       7
                                       6
                                     255.8
                                     12.6
                                       6
                                 271.1* (↑6)
                                     10.5
                                       6
                                     258.3
                                      9.5
                                       6
                                     249.8
                                      7.5
                                      10
                                       6
                                     271.9
                                     11.2
                                       6
                                292.3** (↑7)
                                     11.7
                                       6
                                     272.4
                                     10.8
                                       6
                                     260.5
                                      8.4
                                 11 (Terminal)
                                       6
                                     275.8
                                     13.2
                                       6
                                298.1** (↑8)
                                     13.0
                                       6
                                     276.6
                                      9.6
                                       6
                                     266.1
                                      8.2
                         Body Weight Gain (Days 1-11)
                                       6
                                     49.9
                                      5.1
                                       6
                                71.5** (↑43)
                                      7.1
                                       6
                                     49.1
                                      5.3
                                       6
                                 41.3* (↓17)
                                      4.3
  a  Data were obtained from  the study report. Percent differences from vehicle control are included in parentheses.
  N =Number of animals in the group
  SD= Standard Deviation
  * Significantly different from controls at p<0.05;
  ** Significantly different from controls at p<0.01

Table 22:  Chemical S - Selected Group Mean (+-SD) Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen Assay [a]
                                  Study Day #
                                     Dose

                               Negative Control
                              TP (0.4 mg/kg/day)
                               Positive Control
                              FT (3 mg/kg/day) +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                               (10 mg/kg/day) +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                               (40 mg/kg/day) +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                               (75 mg/kg/day) +
                              TP (0.4 mg/kg/day)

                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       N
                                     Mean
                                      SD
                                       1
                                       6
                                     208.4
                                      8.6
                                       6
                                     209.6
                                      9.7
                                       6
                                     207.1
                                      9.4
                                       6
                                     209.1
                                      7.9
                                       6
                                     211.0
                                      7.5
                                       4
                                       6
                                     232.6
                                      6.3
                                       6
                                     227.0
                                      7.5
                                       6
                                     230.5
                                      7.9
                                       6
                                     224.9
                                      7.7
                                       6
                                 215.0* (↓8)
                                     12.9
                                       5
                                       6
                                     237.7
                                      7.4
                                       6
                                     232.6
                                      8.3
                                       6
                                     236.8
                                      7.7
                                       6
                                     227.8
                                      8.3
                                       6
                                 221.6* (↓7)
                                     13.6
                                       7
                                       6
                                     252.8
                                      6.2
                                       6
                                     243.2
                                      9.5
                                       6
                                     248.4
                                      6.4
                                       6
                                 238.9* (↓5)
                                      7.1
                                       6
                                229.4** (↓9)
                                     14.1
                                      10
                                       6
                                     272.6
                                      8.1
                                       6
                                     261.0
                                      8.2
                                       6
                                     268.8
                                      6.6
                                       6
                                 255.4* (↓6)
                                      7.7
                                       6
                                245.2** (↓10)
                                     14.0
                                 11 (Terminal)
                                       6
                                     277.2
                                      9.4
                                       6
                                     267.2
                                      9.0
                                       6
                                     273.7
                                      6.8
                                       6
                                257.9** (↓7)
                                      6.5
                                       6
                                248.8** (↓10)
                                     15.2
                         Body Weight Gain (Days 1-11)
                                       6
                                     68.8
                                     12.1
                                       6
                                     57.5
                                      2.1
                                       6
                                     66.5
                                      6.0
                                       6
                                48.7** (↓29)
                                      4.2
                                       6
                                37.8** (↓45)
                                     11.9
a Data were obtained from the study report.  Percent differences from negative control  are included in parentheses.
N=Number of animals in the group
SD=Standard Deviation
 * Significantly different from controls at p<0.05
 ** Significantly different from controls at p<0.01

		Table 23:  Chemical S - Group Mean (+-SD) Overall (Days 1-11) Food Consumption (g/rat/day) [a]
                                     Dose
                                       N
                                     Mean
                                      SD
                            Androgen Agonist Phase
Vehicle control (corn oil; 0)
                                       3
                                     24.76
                                     0.38
Positive control (TP; 0.4 mg/kg/day)
                                       3
                                27.96**
(↑13)
                                     0.35
Chemical S (10 mg/kg/day)
                                       3
                                     24.35
                                     0.27
Chemical S (75 mg/kg/day)
                                       3
                                23.38**
(↓6)
                                     0.35
                              Anti-Androgen Phase
Negative control (TP; 0.4 mg/kg/day)
                                       3
                                     23.87
                                     0.38
Positive control (FT+TP; 3 mg/kg/day +0.4 mg/kg/day)
                                       3
                                20.69**
(↓13)
                                     0.58
Chemical S+TP (10 mg/kg/day +0.4 mg/kg/day)
                                       3
                                21.13**
(↓11)
                                     0.58
Chemical S+TP (40 mg/kg/day +0.4 mg/kg/day)
                                       3
                                18.05**
(↓24)
                                     0.50
Chemical S+TP (75 mg/kg/day +0.4 mg/kg/day)
                                       3
                                15.89**
(↓33)
                                     0.50
a Data were obtained from the study report.  Percent differences from vehicle or negative controls are included in parentheses.
N=Number of observations
SD=Standard Deviation
** Significantly different from controls at p<0.01

Table 24:  Chemical S - Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Androgen Agonist Assay in Rats [a]
                                     Organ
                                     Dose

                                Vehicle Control
                               Positive Control
                              TP (0.4 mg/kg/day)
                           Chemical S (10 mg/kg/day)
                                  Chemical S
                                (75 mg/kg/day)

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
Liver (g)
                                       6
                                     12.24
                                     0.47
                                      3.8
                                       6
                                    13.66**
                                    (↑12)
                                     0.89
                                      6.5
                                       6
                                     12.11
                                     0.60
                                      4.9
                                       6
                                     12.86
                                     0.57
                                      4.4
Kidneys (g)
                                       6
                                     1.860
                                     0.17
                                      9.1
                                       6
                                     2.063
                                     0.07
                                      3.4
                                       6
                                     1.819
                                     0.10
                                      5.6
                                       6
                                     1.722
                                     0.10
                                     5.38
Adrenals (mg)
                                       6
                                     70.92
                                     10.9
                                     15.4
                                       6
                                     68.70
                                     4.79
                                      7.0
                                       6
                                     74.63
                                     13.07
                                     17.5
                                       6
                                     77.35
                                     8.01
                                     10.4
Seminal vesicles (mg)
                                       6
                                     51.7
                                      8.3
                                     16.1
                                       6
                               618.7** (↑1097)
                                     103.9
                                     16.8
                                       6
                                 62.7* (↑21)
                                      2.6
                                      4.2
                                       6
                                     62.2
                                      9.1
                                     14.7
Ventral prostate (mg)
                                       6
                                     18.8
                                      3.6
                                     18.9
                                       6
                               166.8** (↑787)
                                     32.2
                                     19.3
                                       6
                                     19.3
                                      3.3
                                     17.1
                                       6
                                     20.1
                                      4.8
                                     24.0
LABC (mg)
                                       6
                                     220.4
                                     36.4
                                     16.5
                                       6
                               472.0** (↑114)
                                     57.1
                                     12.1
                                       6
                                     220.3
                                     48.0
                                     21.8
                                       6
                                     187.1
                                     24.5
                                     13.1
Cowper's glands (mg)
                                       6
                                     6.55
                                     2.61
                                     39.8
                                       6
                               42.65** (↑551)
                                     10.4
                                     24.4
                                       6
                                      8.0
                                      2.4
                                     30.1
                                       6
                                      6.2
                                      1.6
                                     26.1
Glans penis (mg)
                                       6
                                     41.5
                                      4.4
                                     10.5
                                       6
                                73.2** (↑76)
                                      5.5
                                      7.6
                                       6
                                     47.3
                                      7.2
                                     15.3
                                       6
                                     45.3
                                      7.5
                                     16.6
	a Data were obtained from the study report.  Percent differences from vehicle controls (calculated by the reviewers) are included in parentheses.
	N=Number of animals in the group
	SD=Standard Deviation
	CV=Coefficient of Variation
 * Significantly different from controls at p<0.05

Table 25:  Chemical S - Weights of Liver, Kidneys, Adrenals and Accessory Sex Organs from Anti-Androgen Assay in Rats [a]
                                     Organ
                               Dose (mg/kg/day)

                               Negative Control
                              TP (0.4 mg/kg/day)
                               Positive Control
                              FT (3 mg/kg/day) +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                               (10 mg/kg/day) +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                               (40 mg/kg/day)  +
                              TP (0.4 mg/kg/day)
                                  Chemical S
                      (75 mg/kg/day) + TP (0.4 mg/kg/day)

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
Liver (g)
                                       6
                                     12.57
                                     1.50
                                     11.9
                                       6
                                     12.17
                                     0.64
                                      5.3
                                       6
                                     13.30
                                     0.60
                                      4.5
                                       6
                                     11.87
                                     0.84
                                      7.0
                                       6
                                     11.82
                                     1.48
                                     12.5
Kidneys (g)
                                       6
                                     1.886
                                     0.15
                                      8.1
                                       6
                                     1.741
                                     0.13
                                      7.6
                                       6
                                     1.842
                                     0.14
                                      7.8
                                       6
                                     1.698
                                     0.15
                                      8.9
                                       6
                                     1.744
                                     0.18
                                     10.4
Adrenals (mg)
                                       6
                                     73.95
                                     4.25
                                      5.8
                                       6
                                     69.60
                                     8.94
                                     12.9
                                       6
                                     63.73
                                     6.78
                                     10.6
                                       6
                                     70.13
                                     6.85
                                      9.8
                                       6
                                     70.15
                                      8.7
                                     12.4
Seminal vesicles (mg)
                                       6
                                     389.8
                                     46.1
                                     11.8
                                       6
                                74.9** (↓81)
                                      7.6
                                     10.2
                                       6
                                     327.3
                                     51.2
                                     15.6
                                       6
                                256.2** (↓34)
                                     69.7
                                     27.2
                                       6
                                302.1** (↓23)
                                     64.6
                                     21.4
Ventral prostate(mg)
                                       6
                                     128.1
                                     21.6
                                     16.9
                                       6
                                36.2** (↓72)
                                      5.3
                                     14.5
                                       6
                                104.0* (↓19)
                                     17.0
                                     16.4
                                       6
                                77.2** (↓40)
                                     14.3
                                     18.6
                                       6
                                97.3** (↓24)
                                     17.3
                                     17.8
LABC (mg)
                                       6
                                     426.5
                                     42.0
                                      9.9
                                       6
                                224.3** (↓47)
                                     22.7
                                     10.1
                                       6
                                     419.6
                                     26.0
                                      6.2
                                       6
                                359.8** (↓16)
                                     34.5
                                      9.6
                                       6
                                326.8** (↓23)
                                     21.4
                                      6.5
Cowper's glands (mg)
                                       6
                                     31.7
                                      3.6
                                     11.3
                                       6
                                10.4** (↓67)
                                      2.5
                                     23.8
                                       6
                                     30.1
                                      7.0
                                     23.4
                                       6
                                23.0** (↓27)
                                      4.7
                                     20.5
                                       6
                                     28.2
                                      7.7
                                     27.3
Glans penis (mg)
                                       6
                                     75.27
                                      8.9
                                     11.8
                                       6
                                49.0** (↓35)
                                      7.2
                                     14.8
                                       6
                                63.4** (↓16)
                                      4.2
                                      6.7
                                       6
                                60.1** (↓20)
                                      3.8
                                      6.3
                                       6
                                     70.5
                                      3.3
                                      4.6
a Data were obtained from the study report.  Percent differences from vehicle controls are included in parentheses.
N=Number of animals in the group
SD=Standard Deviation
CV=Coefficient of Variation
*Significantly different from controls at p<0.05
**Significantly different from controls at p<0.01

              vii. Pubertal Female Assay (Rat)
In a Female Pubertal Assay, 15 Wistar rats/dose group were treated daily via oral gavage with Chemical S (97.85% a.i) in corn oil at doses of 0, 10, or 75 mg/kg bw/day from post-natal day (PND) 22 to 42.  An additional positive control group of 15 rats was similarly treated with 1chloro-2-nitrobenzene in corn oil daily via oral gavage at a dose of 100 mg/kg bw/day.  Animals were examined daily for vaginal opening (VO) beginning on PND 22; age and body weight at day of attainment were also recorded.  Following sacrifice of the rats on PND 42, blood was collected for clinical chemistry analyses, including selected hormones.  Total serum thyroxine (T4) and thyroid stimulating hormone (TSH) levels were determined using the enzyme-linked immunosorbent assay (ELISA).  Liver, adrenals, pituitary, thyroid, and urogenital organs were collected and weighed.  Histology examinations were performed on the uterus, thyroid, one ovary, one kidney, liver, pituitary, adrenals, and gross lesions.  Summary data are presented in Tables 26 through 31.
No treatment-related effects were observed on mortality.  Thyroid colloid area and follicular cell height scores and the incidence of gross or microscopic lesions were similar in the treated groups and the vehicle controls.  The considerable delay in onset of vaginal opening in the 75 mg/kg bw/day group (PND 38; sacrifice at PND 42) precluded the proper evaluation of the estrous cycle and of mean age at first vaginal estrus.
 At 10 mg/kg bw/day, unadjusted and adjusted ages at VO were increased (p<0.01) by 2.1-2.9 days.  The adjusted body weight at VO was increased (p<0.05) by 7%.  Mean cycle length was reduced to 3.5 days compared to 5.6 days in the vehicle control.  The number of females cycling was increased to 80% compared to the vehicle control (67%), but the number of females regularly cycling was 67% (p<0.05) in the treated group compared to 80% in the vehicle control.  It was unclear if there was an effect on estrous cyclicity.  One animal in this group had a distended cervix; however, this finding was considered incidental.  Primordial follicular cell counts were decreased (p<0.05) by 39% at 10 mg/kg/day, but this effect was not noted at 75 mg/kg bw/day.
At 75 mg/kg/day, increased incidences (# affected/# exposed) of slight to moderate salivation were observed post-dose (15/15) compared to 0/15 at 0 or 10 mg/kg bw/day.  Body weights were decreased (p<0.01) by 8-20% from PND 24 to 42 compared to vehicle controls.  Adjusted and unadjusted final body weights were decreased (p<0.01) by 13% each, and adjusted and unadjusted overall body weight gains (PND 22-42) were decreased (p<0.01) by 20% each.  Food consumption was decreased (p<0.01) by 27 − 37% during the measured intervals from PND 22-36.  At 75 mg/kg bw/day, serum glucose was slightly decreased (p<0.05) by 14%, which was considered incidental.  Minor decreases (p<0.05; within historical control range) were observed in serum total protein and serum albumin (↓8-10%).
At 75 mg/kg bw/day, the following decreases (p<0.01; unless indicated otherwise) in unadjusted and adjusted organ weights were observed compared to vehicle controls (% decrease):  (i) ovaries (15-43%); (ii) wet uterus (24% each; not statistically significant [NS]); (iii) blotted uterus (39% each); and (iv) thyroid (14% each).  Additionally, decreases (p<0.01) were noted in unadjusted, adjusted, and relative kidney weights (9-21%) and pituitary weights (26-36%).  A minor increase (p<0.01) of 11% was noted in the relative liver weight.  Reduced uterus size was observed (7/15) compared to 0 animals in the vehicle control.  The adjusted body weight at VO was increased (p<0.05) by 12%.  Unadjusted and adjusted ages at VO were increased (p<0.01) by 6.5-7.3 days.   In addition, the proportion of 75 mg/kg bw/day females that did not attain VO was 12/15, compared to 0/15 vehicle controls.
At 75 mg/kg bw/day, increased incidences (# affected/15) of the following microscopic findings were noted compared to the vehicle controls (0-1/15):  absent corpus luteum in the ovaries (13), uterine luminal dilation (4), and uterine atrophy (9).
Chemical S significantly altered pubertal development at 75 mg/kg, a dose that had a number of overtly toxic indications.  However, there were signs of delayed puberty in the lower dose as well in the absence of overt toxicity.
In the positive control group, slight to moderate salivation was observed (9/15).  Body weights were decreased (p<0.05) by 6-9% during PND 24-33; however, body weights were similar to the vehicle control thereafter.  Food consumption was decreased (p<0.01) by 21% during the measured interval of PND 22-29.  Unadjusted and adjusted ages at incomplete VO were increased (p<0.05) by 3.7 days, and unadjusted and adjusted ages at VO were increased (p<0.01) by 3.0-3.8 days.  The following differences (p<0.05) were observed in organ weights:  (i) unadjusted, adjusted, and relative liver weights increased by 51-54%; (ii) relative kidney weight increased by 5%; and (iii) adjusted pituitary weight decreased by 13%.  The number of females regularly cycling was only 33% (p<0.05) compared to controls (80%).
The following differences (p<0.01) were noted in clinical chemistry parameters:  glucose (↓14%), total bilirubin (↑471%), and cholesterol (↑21%).  Spleen enlargement (5/15) was noted compared to 0/15 in the other groups.  Increased incidences (# affected/15) of the following microscopic findings were noted compared to the vehicle controls (0-1/15):  adrenal accessory cortical tissue (3), spleen sinusoidal congestion (11), and spleen extramedullary hematopoiesis (11).  Primordial follicular cell counts were decreased (p<0.05) by 35%.
Chemical S was positive for E pathway in the absence of overt toxicity and negative for T pathway in the pubertal female assay.

Table 26:  Selected Body Weight of Female Rats Treated with Chemical S in the Female Pubertal Assay a
                                      PND
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                      22
                                      15
                                     42.07
                                     3.03
                                      7.2
                                      15
                                     42.13
                                     2.33
                                      5.5
                                      15
                                     42.39
                                     2.96
                                      7.0
                                      15
                                     42.35
                                     2.75
                                      6.5
                                      24
                                      15
                                     48.42
                                     3.46
                                      7.2
                                      15
                                     48.94
                                     3.10
                                      6.3
                                      15
                                44.73** (↓8)
                                     3.12
                                      7.0
                                      15
                                 45.47* (↓6)
                                     2.92
                                      6.4
                                      28
                                      15
                                     64.34
                                     4.67
                                      7.3
                                      15
                                     64.17
                                     3.62
                                      5.6
                                      15
                                55.55** (↓14)
                                     4.06
                                      7.3
                                      15
                                58.87** (↓9)
                                     4.73
                                      8.0
                                      36
                                      15
                                     99.79
                                     7.70
                                      7.7
                                      15
                                     98.51
                                     6.11
                                      6.2
                                      15
                                80.11** (↓20)
                                     8.41
                                     10.5
                                      15
                                     94.96
                                     7.69
                                      8.1
                                      42
                                      15
                                    122.78
                                     9.33
                                      7.6
                                      15
                                    119.72
                                     7.57
                                      6.3
                                      15
                               107.08** (↓13)
                                     11.27
                                     10.5
                                      15
                                    120.68
                                     9.21
                                      7.6
a	Data were obtained from the study report, and %CV was rounded by the reviewers.  Percent difference from control (calculated by reviewers from unrounded cited data) is included in parentheses.
b	100 mg/kg bw/day 1-Chloro-2-Nitrobenzene
N	Number of animals examined
SD	Standard Deviation
CV	Coefficient of Variation
*	Significantly different from controls at p<0.05.
**	Significantly different from controls at p<0.01.

Table 27:  Selected Food Consumption Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a
                                      PND
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                     22-29
                                       8
                                     9.27
                                     0.48
                                      5.2
                                       8
                                     8.79
                                     0.36
                                      4.1
                                       8
                                5.82** (↓37)
                                     0.97
                                     16.7
                                       8
                                7.29** (↓21)
                                     0.63
                                      8.7
                                     29-36
                                       8
                                     13.48
                                     1.04
                                      7.7
                                       8
                                     12.74
                                     0.93
                                      7.3
                                       8
                                9.84** (↓27)
                                     1.97
                                     20.0
                                       8
                                     12.56
                                     1.30
                                     10.4
                                     36-40
                                       8
                                     15.79
                                     1.30
                                      8.2
                                       8
                                     15.31
                                     1.11
                                      7.3
                                       8
                                     13.06
                                     2.82
                                     21.6
                                       8
                                     15.42
                                     1.27
                                      8.3
                                     40-42
                                       8
                                     14.93
                                     1.11
                                     7.46
                                       8
                                     14.72
                                     1.17
                                     7.93
                                       8
                                     14.34
                                     2.35
                                     16.4
                                       8
                                     16.08
                                     1.55
                                      9.6
a Data were obtained from the study report, and %CV was rounded by the reviewers.  Percent difference from control (calculated by reviewers from unrounded cited data) is included in parentheses.
b 100 mg/kg bw/day 1-Chloro-2-Nitrobenzene
N=Number of animals examined
SD=Standard Deviation
CV=Coefficient of Variation
**Significantly different from controls at p<0.01.

Table 28: General Growth and Vaginal Opening of Rats Treated with Chemical S in the Female Pubertal Assay a
                              Parameter Evaluated
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
Initial Body Weight
(PND 22; g)
                                       U
                                      15
                                     42.1
                                     3.03
                                      7.2
                                      15
                                     42.1
                                     2.33
                                      5.5
                                      15
                                     42.4
                                     2.96
                                      7.0
                                      15
                                     42.4
                                     2.75
                                      6.5

                                       A
                                      15
                                     42.2
                                     0.63
                                      1.5
                                      15
                                     42.2
                                     0.63
                                      1.5
                                      15
                                     42.3
                                     0.63
                                      1.5
                                      15
                                     42.3
                                     0.63
                                      1.5
Body Weight at VO (g)
                                       U
                                      15
                                     98.3
                                     7.29
                                      7.4
                                      13
                                     104.8
                                     8.04
                                      7.7
                                       3
                                     110.3
                                     5.13
                                      4.7
                                      12
                                     107.7
                                     13.6
                                     12.6

                                       A
                                      15
                                     98.4
                                     9.24
                                      9.4
                                      13
                                 105.2* (↑7)
                                     9.26
                                      8.8
                                       3
                                110.1* (↑12)
                                     9.24
                                      8.4
                                      12
                                 107.2* (↑9)
                                     9.27
                                      8.7
Final Body Weight
(PND 42; g)
                                       U
                                      15
                                     122.8
                                     9.33
                                      7.6
                                      15
                                     119.7
                                     7.57
                                      6.3
                                      15
                                107.1** (↓13)
                                     11.27
                                     10.5
                                      15
                                     120.7
                                     9.21
                                      7.6

                                       A
                                      15
                                     122.9
                                     4.23
                                      3.4
                                      15
                                     119.9
                                     4.23
                                      3.5
                                      15
                                106.9** (↓13)
                                     4.23
                                      4.0
                                      15
                                     120.6
                                     4.23
                                      3.5
Final Body Weight
(% of control)
                                       U
                                      NA
                                     97.5
                                     87.2
                                     98.3

                                       A
                                      NA
                                     97.5
                                     87.0
                                     98.1
Body Weight Gain (PND 22-42; g)
                                       U
                                      15
                                     80.7
                                     8.25
                                     10.2
                                      15
                                     77.6
                                     6.74
                                      8.7
                                      15
                                64.7** (↓20)
                                     10.75
                                     16.6
                                      15
                                     78.3
                                     7.71
                                      9.9

                                       A
                                      15
                                     80.8
                                     4.26
                                      5.3
                                      15
                                     77.7
                                     4.26
                                      5.5
                                      15
                                64.6** (↓20)
                                     4.26
                                      6.6
                                      15
                                     78.3
                                     4.26
                                      5.4
Age at Incomplete VO (PND)
                                       U
                                      15
                                     32.5
                                     2.10
                                      6.5
                                      14
                                     33.4
                                     1.16
                                      3.5
                                       7
                                     38.3
                                     3.15
                                      8.2
                                      13
                                 36.2* (↑12)
                                     1.92
                                      5.3

                                       A
                                      15
                                     32.5
                                     1.91
                                      5.9
                                      14
                                     33.4
                                     1.92
                                      5.8
                                       6
                                     39.1
                                     1.93
                                      4.9
                                      13
                                36.2** (↑12)
                                     1.91
                                      5.3
Age at Complete VO (PND)
                                       U
                                      15
                                     35.5
                                     1.25
                                      3.5
                                      15
                                 38.4** (↑8)
                                     2.56
                                      6.7
                                      15
                                42.8** (↑20)
                                     0.41
                                      1.0
                                      15
                                39.3** (↑11)
                                     2.53
                                      6.4

                                       A
                                      15
                                     35.5
                                     1.59
                                      4.5
                                      13
                                 37.6** (↑6)
                                     1.60
                                      4.2
                                       3
                                42.0** (↑18)
                                     1.59
                                      3.8
                                      12
                                 38.5** (↑8)
                                     1.60
                                      4.2
Proportion unopened (#/N)
                                     0/15
                                     2/15
                                     12/15
                                     3/15

a	Data were obtained from the study report, and were sometimes rounded by the reviewers.  Percent difference from control is included in parentheses.
b	100 mg 1-Chloro-2-Nitrobenzene
U	Unadjusted for body weight on PND 21;  A	Adjusted for body weight on PND 21
N	Number of animals examined; SD	Standard Deviation;  CV	Coefficient of Variation;  NA	Not applicable
*	Significantly different from controls at p<0.05;**	Significantly different from controls at p<0.01
Table 29:  Organ Weight Data of Female Rats Treated with Chemical S in the Female Pubertal Assay a
                                     Organ
                                Vehicle Control
                                  Chemical S
                                10 mg/kg bw/day
                                  Chemical S
                                75 mg/kg bw/day
                              Positive Control b

                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
                                       N
                                     Mean
                                      SD
                                    CV (%)
Liver (g)
                                       U
                                      15
                                     5.72
                                     0.63
                                     11.0
                                      15
                                     5.49
                                     0.58
                                     10.5
                                      15
                                     5.57
                                     0.98
                                     17.6
                                      15
                                8.65** (↑51)
                                     0.96
                                     11.1

                                       A
                                      15
                                     5.73
                                     0.40
                                      7.0
                                      15
                                     5.49
                                     0.40
                                     7.33
                                      15
                                     5.56
                                     0.40
                                     7.25
                                      15
                                8.65** (↑51)
                                     0.40
                                      4.7

                                       R
                                      15
                                     4.66
                                     0.34
                                      7.2
                                      15
                                     4.58
                                     0.31
                                      6.7
                                      15
                                5.17** (↑11)
                                     0.50
                                      9.8
                                      15
                                7.16** (↑54)
                                     0.40
                                      5.5
Kidneys (g)
                                       U
                                      15
                                     0.95
                                     0.12
                                     12.6
                                      15
                                     0.90
                                     0.07
                                      7.2
                                      15
                                0.75** (↓21)
                                     0.09
                                     11.6
                                      15
                                     0.98
                                     0.10
                                     10.4

                                       A
                                      15
                                     0.95
                                     0.04
                                      4.6
                                      15
                                     0.91
                                     0.04
                                      4.9
                                      15
                                0.75** (↓21)
                                     0.04
                                      5.9
                                      15
                                     0.98
                                     0.04
                                      4.5

                                       R
                                      15
                                     0.77
                                     0.05
                                      6.8
                                      15
                                     0.76
                                     0.04
                                      5.8
                                      15
                                 0.70** (↓9)
                                     0.03
                                     3.65
                                      15
                                 0.81* (↑5)
                                     0.04
                                      5.3
Pituitary (mg)
                                       U
                                      15
                                     5.19
                                     0.98
                                     18.9
                                      15
                                     4.73
                                     0.65
                                     13.8
                                      15
                                3.35** (↓35)
                                     0.76
                                     22.6
                                      15
                                     4.55
                                     0.95
                                     20.9

                                       A
                                      15
                                     5.20
                                     0.41
                                      7.8
                                      15
                                     4.74
                                     0.41
                                      8.6
                                      15
                                3.33** (↓36)
                                     0.41
                                     12.2
                                      15
                                 4.55* (↓13)
                                     0.41
                                      8.9

                                       R
                                      15
                                     0.004
                                     0.001
                                     16.1
                                      15
                                     0.004
                                     0.001
                                     15.0
                                      15
                                0.003** (↓26)
                                     0.001
                                     19.7
                                      15
                                     0.004
                                     0.001
                                     17.4
Adrenals (mg)
                                       U
                                      15
                                     38.17
                                     7.15