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TOXICOLOGICAL SCIENCES 69, 92–108 (2002) Copyright © 2002 by the Society of Toxicology

Evaluation of a 15-Day Screening Assay Using Intact Male Rats for Identifying Antiandrogens John C. O’Connor, 1 Steven R. Frame, and Gregory S. Ladics DuPont Haskell Laboratory for Health and Environmental Sciences, P.O. Box 50, Newark, Delaware 19714 Received March 18, 2002; accepted June 7, 2002

primary humoral immune response to SRBC, spleen or thymus weights, or spleen cell number. In the current study, 5 of the six test substances were identified as endocrine-active substances consistent with their known/proposed mechanism(s) of action. The effects that were observed in the current study via oral (gavage) compound administration were similar to the responses that were observed by the ip route in previous studies for DDE and FLUT. This report, in addition to the > 20 compounds that have already been examined using the 15-day intact male assay, supports this assay as a viable screening assay for detecting EACs, and also illustrates that the ability to identify EACs using the intact male assay will be equivalent regardless of the route of compound administration. Key Words: screening; Tier I battery; rats; immunotoxicity; endocrine-active compounds; p,pⴕ-DDE; cyproterone acetate; flutamide; linuron; vinclozolin; di-n-butyl phthalate.

An in vivo screening assay using intact adult male rats has been evaluated for its ability to detect six antiandrogenic compounds via oral administration. The test compounds included cyproterone acetate (CPA), flutamide (FLUT), p,pⴕ-DDE (DDE), di-n-butyl phthalate (DBP), linuron (LIN), and vinclozolin (VCZ). Two of the test compounds (DDE and FLUT) have been previously evaluated in the 15-day intact male assay with compound administration via intraperitoneal injection (ip). For the current studies, male rats were dosed for 15 days via oral gavage and euthanized on the morning of test day 15. The endpoints evaluated included final body and organ weights (liver, thyroid gland, testes, epididymides, prostate, seminal vesicles with fluid, accessory sex gland unit [ASG]), serum hormone concentrations (testosterone [T], estradiol [E2], dihydrotestosterone [DHT], luteinizing hormone [LH], follicle stimulating hormone [FSH], prolactin [PRL], T 3, T 4, and thyroid stimulating hormone[TSH]), and histopathology of the testis, epididymis, and thyroid gland; positive results for each endpoint are described below. In addition, an evaluation of immune system endpoints (humoral immune function, spleen and thymus weights, and spleen cell number) was conducted on a subset of animals dosed with either DDE or FLUT. All six endocrine-active compounds (EACs) increased relative liver weight. FLUT and VCZ caused the typical pattern for an androgen receptor (AR) antagonist, although not all endpoints were statistically significant for VCZ: decreased ASG weights, hormonal alterations (increased T, DHT, LH, and FSH), and induced Leydig cell hypertrophy and/or hyperplasia. CPA caused effects consistent with its mixed AR antagonist/progesterone receptor agonist activity: it decreased ASG weights, caused hormonal alterations (increased T and E2; decreased FSH), and caused spermatid retention. DBP, a compound with antiandrogen-like activity via a nonreceptor mediated mechanism, caused hormonal alterations (decreased T, DHT, and E2; increased LH, FSH, and PRL) and induced general testicular degeneration. LIN, a weak AR antagonist, decreased ASG weights, caused hormonal alterations (decreased T, DHT, and LH; increased E2), and caused spermatid retention. Unlike the other AR antagonists evaluated, DDE, a weak AR antagonist, did not alter reproductive parameters. All six antiandrogens caused some effects on thyroid parameters, although only CPA, DDE, and VCZ caused results consistent with a potential thyroid-modulator. FLUT and DDE did not alter the

Responding to concerns that endocrine-active compounds (EACs) may impact endocrine function in humans and wildlife, Congress passed legislation in 1996 requiring the U.S. Environmental Protection Agency (U.S. EPA) to implement screening/testing strategies for EACs. Subsequently, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen receptors (ER) or androgen receptors (AR), steroid biosynthesis inhibitors, or their ability to alter thyroid function (EDSTAC, 1998). For Tier I, EDSTAC recommended three potential screening batteries comprised of a number of in vitro and in vivo assays (Table 1). The current report examines the utility of one of the proposed screening assays, the 15-day intact male assay, for its ability to identify EACs. The rationale for the study design (Cook et al., 1997) and the pros and cons of this approach have been previously discussed (O’Connor et al., in press). Similar feasibility studies are currently underway in a number of laboratories for the 15-day intact male assay, as well as the other assays that were proposed by EDSTAC. Historically, the Hershberger assay, which utilizes organ

1

To whom correspondence should be addressed. Fax: (302) 366-5003. E-mail: [email protected]. 92

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SCREENING FOR ANTIANDROGENS

TABLE 1 Comparison of the EDSTAC-Recommended and Alternative Tier I Screening Batteries for Identifying Endocrine-Active Compounds Recommended screening battery In vitro assays ER binding/transactivation AR binding/transactivation Minced testis assay for steroidogenesis In vivo mammalian assays Uterotrophic assay (3-day exposure) Hershberger (7–10 day exposure) Pubertal female (20-day exposure) Environmental assays Frog metamorphosis Fish gonadal recrudescence

Alternate screening battery no. 1

Alternate screening battery no. 2

ER binding/transactivation AR binding/transactivation Placental aromatase a

ER binding/transactivation AR binding/transactivation Placental aromatase a

Uterotrophic assay (5-day exposure) Intact adult male (15-day exposure)

Uterotrophic assay (3-day exposure) Pubertal male (20-day exposure)

Frog metamorphosis Fish gonadal recrudescence

Frog metamorphosis Fish gonadal recrudescence

Note. The table shows the endocrine-screening batteries that have been recommended to the U.S. Environmental Protection Agency by the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) as potential approaches for screening for endocrine-active compounds. These 3 potential batteries are currently being considered for validation. Assays that differ between the three potential screening batteries are in italics. Note that the 3-day and 5-day uterotrophic assays are essentially the same model except for the duration and could be used interchangeably. ER, estrogen receptor; AR, androgen receptor. a The placental aromatase assay was included to aid in the detection of aromatase inhibitors. However, alternate 1 is capable of detecting aromatase inhibitors, therefore, the placental aromatase assay would not be required for alternate 1. It is unclear whether alternate 2 would require the inclusion of the placental aromatase assay.

weight measurements from sexually immature rats, has been the preferred assay for detecting weak AR antagonists (Dorfman, 1969a,b; EDSTAC, 1998; Hershberger et al., 1953). In contrast, the intact male assay uses intact adult male rats to detect androgenic compounds. Concerns have been raised within the scientific community regarding the sensitivity of adult animals for detecting compounds with weak antiandrogenic activity. Homeostatic control mechanisms (i.e., positive/ negative feedback loops) in the endocrine system of the sexually mature rat result in attenuated responses on compoundinduced organ weight changes. For example, using RU-23908, sexually mature rats were approximately 7- to 8-fold less sensitive in detecting decreases in seminal vesicle and ventral prostate weights than sexually immature rats (Raynaud et al., 1984). Similar age-dependent sensitivity differences in organ weights have also been observed after exposure to the AR antagonists 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p⬘DDE [DDE]), flutamide (FLUT), and linuron (LIN; Cook et al., 1993; Kelce et al., 1995; O’Connor et al., 1998a; ViguierMartinez et al., 1983a,b). However, the intact feedback mechanisms of the sexually mature rat can also be advantageous for detecting EACs since changes in hormonal patterns can signify very specific xenobiotic challenges to the body. Cook and coworkers have used the AR antagonists FLUT and LIN to demonstrate that sexually mature rats are more sensitive than sexually immature rats for detecting compound-induced hormonal changes (Cook et al., 1993). Using a study design similar to Viguier-Martinez and coworkers (1983a,b), LINinduced changes in male reproductive organ weights were greater in immature rats than in mature rats, where decreases in accessory sex gland (ASG) unit weight were approximately

3-fold greater than in sexually mature rats (Cook et al., 1993). In contrast, LIN-induced changes in serum hormone levels, consistent with those of an AR antagonist, were observed in sexually mature rats but not in sexually immature rats (Cook et al., 1993). These data show that weak AR antagonists such as LIN can be detected using an adult rat model, and illustrate the utility of the comprehensive hormonal assessment that is included in the 15-day intact male assay. Additionally, there is increasing concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect not only the endocrine and reproductive systems but also the immune system (Crisp et al., 1997; Kavlock et al., 1996). A highly complex interrelationship exists between the immune and neuroendocrine systems (reviewed in Besedovsky and del Rey, 1996; Chryssikopoulos, 1997; Fuchs and Sanders, 1994; Tomaszewska and Przekop, 1997). Immune cells have been reported to produce various peptide and protein hormones (e.g., growth hormone, prolactin, lutenizing hormone, thyrotropin-stimulating hormone, and adrenocorticotropin; Blalock, 1989; Gaillard, 1995). Furthermore, a number of hormones can suppress, attenuate, or enhance immune system responses (Gaillard, 1995; Weigent and Blalock, 1987), while cytokines produced by the immune system can alter neuroendocrine functions (Rivest and Laflamme, 1995; Roy, 1994). In the current study, six antiandrogens with a wide range of potencies have been examined using the 15-day intact male assay with compound administration via the oral (gavage) route. The readers are also referred to the accompanying article (O’Connor et al., 2002), which presents data from four additional EACs that were evaluated via oral compound adminis-

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tration in the 15-day intact male assay. These studies will provide a basis for comparing differences in sensitivity between the ip and oral routes of compound administration for the 15-day intact male assay, as well as increasing the number of compounds examined using the intact male assay. In addition to the endocrine endpoints, an evaluation of immune system endpoints was conducted on DDE and FLUT using a separate subset of animals. Spleen and thymus weights, spleen cell number, and the primary IgM humoral immune response to sheep red blood cells (SRBC) were evaluated. Since all six compounds in the current report are antiandrogens, DDE and FLUT were used as model compounds for other antiandrogens for the potential effects on the immune system. Finally, the data from the current report were compared to data from the Hershberger and pubertal male assays, two additional screening assays proposed by EDTAC, in order to facilitate comparisons of the three screening assays for their ability to detect antiandrogens. The test compounds that were examined included the antiandrogens cyproterone acetate (CPA; Neri, 1976), DDE (Kelce et al., 1995), di-n-butyl phthalate (DBP; Foster et al., 2001; Mylchreest et al., 1998, 1999, 2000), FLUT (Neri, 1976), LIN (Cook et al., 1993; Lambright et al., 2000), and vinclozolin (VCZ; Gray et al., 1994; VanRavenzwaay, 1992). MATERIALS AND METHODS Test materials. CPA, FLUT, and cyclophosphamide (CY) were obtained from the Sigma Chemical Company (St. Louis, MO). All other materials were obtained from the following manufacturers: Certified Rodent Diet #5002威, PMI Feeds, Inc. (St. Louis, MO); DDE (Catalog No. 12,389-7) (2,2-bis(4chlorophenyl)-1,1-dichloroethylene), Aldrich Chemical Company (Milwaukee, WI); methylcellulose, Fisher Scientific (Springfield, NJ); LIN and VCZ, Chem Service (West Chester, PA); sterile sheep red blood cell (SRBC) in Alsevers solution, Rockland (Gilbertsville, PA); Hanks Balanced Salt Solution (HBSS) and 1.0 M HEPES, GIBCO Laboratories (Grand Island, NY); luteinizing hormone (LH; catalog #RPA.552), prolactin (PRL; catalog #RPA.553), thyroid stimulating hormone (TSH; catalog #RPA.554), and follicle stimulating hormone (FSH; catalog #RPA.550) radioimmunoassay (RIA) kits, Amersham Corp. (Arlington Heights, IL); testosterone (T; catalog #TKTT5), estradiol (E2; catalog #KE2D5), tri-iodothyronine (T 3; catalog #TKT35), and thyroxine (T 4; catalog #TKT45) RIA kits, Diagnostic Products Corp. (Los Angeles, CA); rT 3 RIA kits (catalog #10834), Polymedco Inc. (Cortlandt Manor, NY); and, dihydrotestosterone (DHT; catalog #DSL-9600) RIA kit, Diagnostic Systems Laboratories (Webster, TX). DBP was generously donated by Eastman Chemical Company (Kingsport, TN). Test species. Male Sprague-Dawley (Crl:CD威(SD)IGS BR) rats were acquired from Charles River Laboratories, Inc. (Raleigh, NC). Male rats were approximately 63-days-old upon arrival. Rats were housed in stainless steel, wire-mesh cages suspended above cage boards and were fed PMI Feeds, Inc., Certified Rodent Diet #5002威 and provided with tap water (United Water Delaware) ad libitum. Rats were clinically normal and free of antibody titers to pathogenic murine viruses and mycoplasma and free of pathogenic endoand ectoparasites and bacteria. Animal rooms were maintained on a 12-h light/dark cycle (fluorescent light), a temperature of 23 ⫾ 2°C, and a relatively humidity of 50 ⫾ 10%. After a quarantine period of approximately one week, rats that displayed adequate weight gain and freedom from clinical signs were divided by computerized, stratified randomization into 5 treatment groups so that there were no statistically significant differences among group body

weight means. For DDE and FLUT, each treatment group contained 25 male rats, 15 were designated for endocrine analyses and 10 were designated for immune system analyses. For CPA, DBP, LIN, and VCZ each treatment group contained 15 male rats designated for endocrine analyses; immune system analyses were not conducted. Study design. Each of the six test compounds were evaluated with their own concurrent control group in individual studies over a period of two years. All rats were weighed daily and cage-side examinations were performed to detect moribund or dead rats. At each weighing, rats were individually handled and examined for abnormal behavior or appearance. No mortality was observed for the six test substances. CPA, DDE, FLUT, LIN, and VCZ were prepared in 0.25% methylcellulose vehicle; DBP was prepared in corn oil vehicle. All test substances were administered by oral gavage at approximately 0900 h daily for 15 days. The dose volume was 5.0 ml/kg body weight for animals dosed with CPA, DDE, FLUT, LIN, and VCZ or 2.0 ml/kg for rats dosed with DBP. Control rats received vehicle only. On the morning of test day ⫹15, rats were anesthetized using CO 2 and euthanized by exsanguination. The following concentrations of test compound were used: CPA (1, 10, 50, and 100 mg/kg/day), DDE (50, 100, 200, and 300 mg/kg/day), DBP (250, 500, 750, and 1000 mg/kg/day), FLUT (5, 20, 50, and 100 mg/kg/day), LIN (25, 50, 100, and 150 mg/kg/day), and VCZ (10, 75, 150, and 300 mg/kg/day). Doses were selected in order to obtain the maximal pharmacologic effect for each compound and/or not exceed the maximum tolerated dose (MTD), defined as a 10% difference in final body weight from the ad libitum control group, as determined in range-finder studies. Pathological evaluations. For the endocrine subset (15/group) all rats were administered the final dose of test compound approximately 2 h prior to euthanization. Euthanization was performed between 0700 and 1000 h on the morning of test day ⫹15, and necropsy was performed across the treatment groups in order to control for potential variation due to “time-of-day” effects. At necropsy, blood was collected from the descending vena cava, allowed to clot at 4°C for 1 h, and centrifuged for 20 min at 1500 ⫻ g (4°C) for preparation of serum for hormonal analyses. The liver, testes, epididymides, prostate, seminal vesicles, and ASG unit (composed of the prostate, seminal vesicles with fluid, and coagulating glands) were weighed and relative (to body weight) organ weights were calculated. The thyroid glands and surrounding tissue were removed and placed into formalin fixative for at least 48 h prior to trimming and weighing. Following fixation, final dissection of the thyroid gland was performed under a dissecting microscope by one individual in order to reduce the variability of the dissection procedure and hence, reduce the variability of the thyroid weights. Weights for the testes, epididymides, and seminal vesicles were paired weights. After weighing, the epididymides were placed in formalin fixative, the testes were placed in Bouin’s fixative, and both were examined microscopically. The formalin-fixed thyroid glands were also examined microscopically. The epididymides were inadvertently not collected from the VCZ experiment. Immune system assessment. For the immune system subset, rats were housed and dosed under the same conditions as the main study animals. Six days prior to study termination, rats dosed with DDE and FLUT that were designated for assessment of the primary humoral immune response (10/dose group) were injected into a tail vein with 0.5 ml of 4 ⫻ 10 8 SRBC in saline. This dose of SRBC, in conjunction with the timing of immunization relative to measurement of response, was found to elicit an optimal primary IgM response in this rat strain (data not shown). All rats were administered the final dose of test compound approximately 2 h prior to euthanization on the morning of test day ⫹15. Between 0800 and 1100 h, rats were euthanized, blood collected, and the spleen and thymus were removed, weighed, and relative (to body weight) organ weights calculated. Sera were obtained and stored frozen (0°C) until analyzed. A single-cell suspension was prepared from half of the spleen in HBSS containing 1.0 M HEPES (GIBCO) by first cutting the spleens into several pieces and then placing the pieces in a Stomacher威 Lab Blender (Seward Medical Limited, London, UK). Spleen cell numbers were determined using a Serono Baker 9000威 hematology analyzer (Allentown, PA) and multiplied by the total spleen weight/weight of the spleen section to obtain cell

SCREENING FOR ANTIANDROGENS number/total spleen. Individual serum samples were analyzed for SRBCspecific IgM antibody using an ELISA as previously described (Temple et al., 1993). Data were acquired using an MR 5000 96-well microplate reader (Dynatech Laboratories, Chantilly, VA) and analyzed using the Revelation Software (Version 2.0, Dynatech Laboratories). Sera pooled from male rats injected with SRBC and dosed with the known immunosuppressive agent CY (20 mg/kg/day, ip) for 6 days were evaluated with the study samples as a positive control. The data were expressed as the highest dilution (i.e., titer) to give an absorbance value of 0.5 (e.g., 1:500 ⫽ 500). The SRBC-specific serum IgM antibody titers were reported as log 2 to normalize the data. Hormonal measurements. Blood was collected at necropsy from all animals, approximately 2 h after the last administered dose. Serum was prepared and stored at ⫺80°C until analyzed for serum hormone concentrations. Serum T, E2, DHT, LH, FSH, PRL, TSH, T 3, and T 4 concentrations were measured by commercially available RIA kits. Details on standard curve concentrations for each RIA kit are available from the manufacturer. When hormone concentrations were below the limit of detection, zero was used as the value for calculations. When hormone concentrations were above the highest standard, the sample was diluted using kit-specific assay “zero calibrator” and reanalyzed. Statistical analyses. Mean final body weights and organ weights were analyzed by a one-way ANOVA. When the corresponding F test for differences among test group means was significant, pairwise comparisons between test and control groups were made with Dunnett’s test (Dunnett, 1955). Bartlett’s test for homogeneity of variances was performed and, when significant (p ⬍ 0.005), was followed by nonparametric procedures (Dunn’s test; Dunn, 1964). Serum hormone concentrations and SRBC-specific IgM levels were analyzed using Jonckheere’s test for trend in a stepdown manner (Hochberg and Tamhane, 1987; Marcus et al., 1976). If a significant dose-response trend was detected, data from the top dose group was excluded and the test repeated until no significant trend was detected. Except for Bartlett’s test, all other significance was judged at p ⬍ 0.05.

RESULTS

Final body and liver weights (Table 2). Mean final body weights were significantly decreased in rats treated with CPA, FLUT, and LIN, with the greatest decreases at the highest dosages (86, 91, and 89% of control, respectively). Mean final body weights were unchanged in rats treated with DDE, DBP, or VCZ. Relative liver weights were significantly increased in rats treated with CPA, DDE, DBP, FLUT, LIN, and VCZ, with the greatest increases at the highest dosage (136, 149, 121, 132, 110, and 121% of control, respectively). Reproductive organ weights and histopathology (Table 2). CPA decreased absolute epididymis weights in a dose-dependent manner, with statistically significant decreases at 10, 50, and 100 mg/kg/day (85, 80, and 75% of control, respectively). Relative ASG unit weights were decreased in a dose-dependent manner and were significantly decreased at 10, 50, and 100 mg/kg/day (60, 41, and 39% of control, respectively). Individual component weights of the ASG were also decreased by CPA. Relative seminal vesicle weights were decreased in a dose-dependent manner and were significantly decreased at all dosages (86, 58, 36, and 34% of control at dosages of 1, 10, 50, and 100 mg/kg/day, respectively). Similarly, relative prostate weights were decreased in a dose-dependent manner and were significantly decreased at 10, 50, and 100 mg/kg/day (67, 55,

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and 51% of control, respectively). Absolute testis weights were not affected by treatment with CPA at the dose levels tested. Microscopically, administration of 50 or 100 mg/kg/day CPA produced slight degeneration and necrosis of pachytene spermatocytes in Stage VII/VIII tubules. Degenerative changes usually occurred in 1–3 individual spermatocytes within a tubule with 5–20 tubules per testis affected. Spermatocyte degeneration occurred in 11/15 and 14/15 animals in the 50 and 100 mg/kg/day groups, respectively. No microscopic changes were present in the testes of rats exposed to 1 or 10 mg/kg/day CPA. There were no compound-related microscopic changes in the epididymides at the dose levels tested. DDE and DBP did not affect any of the reproductive organ weights at the dose levels tested. For DDE, no microscopic changes were present in the testes or epididymides at the dose levels tested. For DBP, bilateral testicular degeneration was present in 6/15 rats administered 1000 mg/kg/day (minimal bilateral testicular degeneration was present in 1/15 each in the control and 750 mg/kg/day groups). In most affected animals in the 1000 mg/kg/day group, testicular changes were very slight and were characterized by disorganization and loss of germ cells within a few, randomly distributed seminiferous tubules. Evidence of testicular germ cell effects were more apparent in the epididymides, which had increased numbers (usually minimal) of sloughed (round) germ cells within epididymal tubules of 10 of 15 rats in the 1000 mg/kg/day group, compared to 1/15 rats in the control group. A slight increase in germ cells was also present in the epididymides of 10/15 rats administered 750 mg/kg/day DBP, even though clear evidence of degeneration was not apparent within seminiferous tubules of the corresponding testes. No compound-related changes were present in the testes or epididymides of rats exposed to ⱕ 500 mg/kg/day DBP. FLUT decreased absolute epididymis weights in a dosedependent manner, with statistically significant decreases at all dosages (86, 81, 71, and 63% of control at 5, 20, 50, and 100 mg/kg/day, respectively). Relative ASG unit weights were decreased in a dose-dependent manner and were significantly decreased at all dosages (78, 61, 41, and 34% of control at 5, 20, 50, and 100 mg/kg/day, respectively). Individual component weights of the ASG were also decreased by FLUT. Relative seminal vesicle weights were decreased in a dosedependent manner and were significantly decreased at 20, 50, and 100 mg/kg/day (62, 39, and 31% of control, respectively). Similarly, relative prostate weights were decreased in a dosedependent manner and were significantly decreased at 20, 50, and 100 mg/kg/day (59, 48, and 43% of control, respectively). Absolute testis weights were not affected by treatment with FLUT at the dose levels tested. Microscopically, minimal diffuse hypertrophy and hyperplasia of interstitial cells was present in all FLUT-treated groups. These changes were present in 7/15, 14/15, 15/15, and 14/15 animals in the 5, 20, 50, and 100 mg/kg/day groups, respectively. Single cell degeneration and necrosis of pachytene spermatocytes in stage VII

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TABLE 2 Intact Male Assay: Final Body Weights and Organ Weights Final BW Dosage Cyproterone acetate 0 1 10 50 100 p,p⬘-DDE 0 50 100 200 300 Di-n-butyl phthalate 0 250 500 750 1000 Flutamide 0 5 20 50 100 Linuron 0 25 50 100 150 Vinclozolin 0 10 75 150 300

Microscopic changes

(g)

(% Control)

Liver

Testes

Epididymides

ASG

Seminal vesicles

Prostate

Testes a

Epididymides b

422 ⫾ 7 387 ⫾ 6* 369 ⫾ 6* 367 ⫾ 5* 362 ⫾ 4*

100 92 87 87 86

3.9 ⫾ 0.1 4.1 ⫾ 0.1 4.2 ⫾ 0.1 5.2 ⫾ 0.1* 5.3 ⫾ 0.1*

3.2 ⫾ 0.1 3.3 ⫾ 0.1 3.2 ⫾ 0.1 3.1 ⫾ 0.1 3.0 ⫾ 0.1

1.16 ⫾ 0.02 1.11 ⫾ 0.02 0.99 ⫾ 0.03* 0.93 ⫾ 0.02* 0.87 ⫾ 0.02*

0.587 ⫾ 0.022 0.518 ⫾ 0.027 0.354 ⫾ 0.022* 0.243 ⫾ 0.010* 0.227 ⫾ 0.017*

0.412 ⫾ 0.017 0.356 ⫾ 0.022** 0.237 ⫾ 0.016** 0.150 ⫾ 0.008** 0.139 ⫾ 0.009**

0.172 ⫾ 0.010 0.156 ⫾ 0.011 0.116 ⫾ 0.009* 0.094 ⫾ 0.005* 0.087 ⫾ 0.009*

0/15 0/15 0/15 11/15 14/15

0/15 –c –c –c 0/15

420 ⫾ 8 430 ⫾ 7 419 ⫾ 9 419 ⫾ 7 421 ⫾ 5

100 102 100 100 100

4.1 ⫾ 0.1 5.0 ⫾ 0.1* 5.1 ⫾ 0.1* 5.7 ⫾ 0.1* 6.1 ⫾ 0.1*

3.4 ⫾ 0.1 3.3 ⫾ 0.1 3.4 ⫾ 0.1 3.4 ⫾ 0.1 d 3.5 ⫾ 0.1

1.28 ⫾ 0.04 1.19 ⫾ 0.03 1.22 ⫾ 0.04 1.25 ⫾ 0.06 d 1.17 ⫾ 0.04

0.602 ⫾ 0.013 0.583 ⫾ 0.014 0.610 ⫾ 0.016 0.632 ⫾ 0.024 0.602 ⫾ 0.019

0.432 ⫾ 0.013 0.430 ⫾ 0.012 0.413 ⫾ 0.021 0.467 ⫾ 0.019 0.426 ⫾ 0.014

0.172 ⫾ 0.009 0.152 ⫾ 0.005 0.198 ⫾ 0.021 0.167 ⫾ 0.008 0.171 ⫾ 0.007

0/15 –c –c –c 0/15

0/15 –c –c –c 0/15

422 ⫾ 7 420 ⫾ 8 410 ⫾ 6 406 ⫾ 6 397 ⫾ 9

100 100 97 96 94

4.2 ⫾ 0.1 4.4 ⫾ 0.1 4.5 ⫾ 0.1* 4.7 ⫾ 0.1* 5.1 ⫾ 0.1*

3.3 ⫾ 0.1 3.3 ⫾ 0.1 3.2 ⫾ 0.1 3.3 ⫾ 0.1 3.2 ⫾ 0.1

1.02 ⫾ 0.03 1.06 ⫾ 0.02 1.04 ⫾ 0.02 1.08 ⫾ 0.02 1.09 ⫾ 0.02

0.532 ⫾ 0.016 0.558 ⫾ 0.016 0.544 ⫾ 0.029 0.537 ⫾ 0.017 0.558 ⫾ 0.018

0.392 ⫾ 0.011 0.403 ⫾ 0.012 0.405 ⫾ 0.026 0.398 ⫾ 0.016 0.417 ⫾ 0.016

0.143 ⫾ 0.006 0.157 ⫾ 0.006 0.148 ⫾ 0.007 0.144 ⫾ 0.008 0.144 ⫾ 0.005

1/15 0/15 0/15 1/15 6/15

1/15 0/15 0/15 10/15 10/15

427 ⫾ 9 423 ⫾ 7 416 ⫾ 9 411 ⫾ 4 390 ⫾ 6*

100 99 97 96 91

3.8 ⫾ 0.1 3.7 ⫾ 0.0 4.1 ⫾ 0.1* 4.5 ⫾ 0.1* 5.0 ⫾ 0.1*

3.3 ⫾ 0.1 3.3 ⫾ 0.1 3.5 ⫾ 0.1 3.4 ⫾ 0.0 3.3 ⫾ 0.1

1.26 ⫾ 0.03 1.08 ⫾ 0.03* 1.02 ⫾ 0.03* 0.90 ⫾ 0.03* 0.79 ⫾ 0.03*

0.588 ⫾ 0.021 0.461 ⫾ 0.027* 0.356 ⫾ 0.014* 0.243 ⫾ 0.016* 0.200 ⫾ 0.011*

0.390 ⫾ 0.025 0.301 ⫾ 0.022 0.240 ⫾ 0.014** 0.151 ⫾ 0.012** 0.119 ⫾ 0.009**

0.190 ⫾ 0.013 0.158 ⫾ 0.011 0.113 ⫾ 0.007* 0.091 ⫾ 0.007* 0.082 ⫾ 0.007*

0/15 7/15 14/15 15/15 14/15

0/15 –c –c –c 0/15

408 ⫾ 8 396 ⫾ 6 391 ⫾ 4 373 ⫾ 6* 362 ⫾ 4*

100 97 96 91 89

3.9 ⫾ 0.0 4.1 ⫾ 0.1* 4.2 ⫾ 0.1* 4.2 ⫾ 0.1* 4.3 ⫾ 0.1*

3.3 ⫾ 0.1 3.4 ⫾ 0.1 3.3 ⫾ 0.1 3.4 ⫾ 0.1 3.3 ⫾ 0.1

1.16 ⫾ 0.03 1.11 ⫾ 0.03 d 1.10 ⫾ 0.02 d 1.07 ⫾ 0.03 1.05 ⫾ 0.03*

0.597 ⫾ 0.016 0.625 ⫾ 0.019 0.623 ⫾ 0.019 0.574 ⫾ 0.017 0.532 ⫾ 0.026*

0.443 ⫾ 0.014 0.482 ⫾ 0.017 0.465 ⫾ 0.016 0.431 ⫾ 0.016 0.407 ⫾ 0.020

0.153 ⫾ 0.008 0.144 ⫾ 0.010 0.160 ⫾ 0.007 0.143 ⫾ 0.006 0.124 ⫾ 0.007*

0/15 0/15 0/15 2/15 4/15

0/15 –c –c –c 0/15

410 ⫾ 7 409 ⫾ 8 403 ⫾ 6 395 ⫾ 7 392 ⫾ 6

100 100 98 96 96

3.9 ⫾ 0.1 4.1 ⫾ 0.1 4.2 ⫾ 0.1 4.5 ⫾ 0.1* 4.7 ⫾ 0.1*

3.1 ⫾ 0.1 3.2 ⫾ 0.1 3.2 ⫾ 0.1 3.2 ⫾ 0.1 3.3 ⫾ 0.0

1.12 ⫾ 0.03 1.01 ⫾ 0.02 1.07 ⫾ 0.03 1.03 ⫾ 0.03** 1.02 ⫾ 0.02**

0.562 ⫾ 0.015 0.538 ⫾ 0.013 0.533 ⫾ 0.025 0.489 ⫾ 0.023 0.474 ⫾ 0.024*

0.387 ⫾ 0.013 0.387 ⫾ 0.010 0.384 ⫾ 0.019 0.338 ⫾ 0.019 0.334 ⫾ 0.019

0.172 ⫾ 0.007 0.148 ⫾ 0.006 0.147 ⫾ 0.007 0.149 ⫾ 0.010 0.138 ⫾ 0.008*

0/15 0/15 0/15 9/15 12/15

–e –e –e –e –e

Note. Values are mean ⫾ SE; n ⫽ 15 unless otherwise noted. Dosage is given in mg/kg/day. BW, body weight; ASG, accessory sex gland unit. Liver, ASG, seminal vesicles, and prostate weights are given as % body weight. Testes and epididymides weights are given in grams. a Incidence of testicular effects. See text for detailed findings. b Incidence of epididymal effects. See text for detailed findings. c Not evaluated since there were no effects in the high dose group. d n ⫽ 14. e Epididymides were not saved for evaluation. *Significantly different (p ⬍ 0.05) from control by Dunnett’s test. **Significantly different (p ⬍ 0.05) from control by Dunn’s test.

tubules was present in 1/15 and 6/15 rats in the 50 and 100 mg/kg/day groups, respectively. Single cell necrosis of spermatcytes was in most instances very slight, most commonly affecting only one to a few spermatocytes within occasional stage VII tubules. LIN decreased epididymis, prostate, and ASG unit weight at the highest dosage (91, 81, and 89% of control, respectively). Absolute testis and relative seminal vesicle weights were not affected by treatment with LIN at the dose levels tested. Microscopically, low incidences of minimal spermatid retention were present in the testes of rats in the 100 and 150

mg/kg/day groups (2/15 and 4/15, respectively). Spermatid retention was characterized by the presence of late step elongate spermatids in stage IX through XII tubules. Retained spermatids were most often observed at the luminal margin but were also present at other levels of the seminiferous epithelium. There were no compound-related microscopic changes in the epididymides at the dose levels tested. VCZ caused a statistically significant decrease in absolute epididymis weights at 150 and 300 mg/kg/day (92 and 91% of control, respectively). Relative ASG unit weights were decreased in a dose-dependent manner and were significantly

SCREENING FOR ANTIANDROGENS

decreased at 300 mg/kg/day (84% of control). Relative prostate weights were numerically decreased at 10, 75, and 150 mg/kg/ day (86, 85, and 87% of control, respectively), and were significantly decreased at 300 mg/kg/day (80% of control). Absolute testis weights and relative seminal vesicle weights were not affected by treatment with VCZ at the dose levels tested, although at 150 and 300 mg/kg/day relative seminal vesicle weights were decreased to 87 and 86% of control, respectively. Microscopically, slight, diffuse hypertrophy of interstitial cells was present in the testes of rats exposed to 150 or 300 mg/kg/day (9/15 and 12/15, respectively). Interstitial cell changes were characterized primarily by increase cytoplasmic area as well as increased cytoplasmic eosinophilia. Qualitatively, interstitial cell number appeared to be increased, particularly in the 300 mg/kg/day group. There were no compound-related microscopic changes in the seminiferous tubules of the testes at the dose levels tested. Reproductive hormone concentrations (Table 3). CPA caused a statistically significant increase in serum T concentrations at 10, 50, and 100 mg/kg/day (163, 167, and 157% of control, respectively). Serum E2 concentrations were significantly increased at all dosages (190, 262, 290, and 287% of control at dosages of 1, 10, 50, and 100 mg/kg/day, respectively). Serum FSH concentrations were significantly decreased at 100 mg/kg/day (79% of control). Serum PRL concentrations were significantly decreased at 10, 50, and 100 mg/kg/day (61, 28, and 26% of control, respectively). Serum LH and DHT concentrations were not affected by CPA treatment at the dose levels tested. DDE increased serum E2 concentrations at the highest dosage (143% of control), and decreased serum DHT concentrations at 100, 200, and 300 mg/kg/day (63, 78, and 65% of control, respectively). Serum T, PRL, LH, and FSH were not affected by DDE treatment at the dose levels tested. DBP caused a statistically significant decrease in serum T concentrations at 500, 750, and 1000 mg/kg/day (68, 73, and 54% of control, respectively). Serum E2 concentrations were significantly decreased at all dosages (34, 13, 10, and 17% of control at 250, 500, 750, and 1000 mg/kg/day, respectively). Although not statistically significant, serum DHT concentrations were decreased at 1000 mg/kg/day (68% of control). Serum LH concentrations were significantly increased at 500, 750, and 1000 mg/kg/day (121, 125, and 125% of control, respectively). Serum FSH concentrations were significantly increased at 1000 mg/kg/day (118% of control). Serum PRL concentrations were significantly decreased at 1000 mg/kg/day (64% of control). FLUT caused a statistically significant increase in serum T (103, 306, 332, and 312% of control at dosages of 5, 20, 50, and 100 mg/kg/day, respectively) and DHT (216, 374, 394, and 326% of control at dosages of 5, 20, 50, and 100 mg/kg/day, respectively) concentrations at all dosages. Serum E2 concentrations were significantly increased at all dosages (195, 218,

97

315, and 407% of control at 5, 20, 50, and 100 mg/kg/day, respectively). Serum FSH (131, 187, 211, and 193% of control at dosages of 5, 20, 50, and 100 mg/kg/day, respectively) and LH (149, 211, 270, and 254% of control at dosages of 5, 20, 50, and 100 mg/kg/day, respectively) concentrations were significantly increased at all dosages. Serum PRL concentrations were not affected by FLUT treatment at the dose levels tested. LIN increased serum E2 concentrations in a dose-dependent manner, with statistically significantly increases at 50, 100, and 150 mg/kg/day (150, 165, and 180% of control, respectively). Serum T and PRL concentrations were significantly decreased at 150 mg/kg/day (60% and 8% of control, respectively). Serum DHT concentrations were decreased at 100 and 150 mg/kg/day (70 and 37% of control, respectively). Serum FSH and LH concentrations were not affected by LIN treatment at the dose levels tested. VCZ caused a statistically significant increase in serum FSH (123, 167, and 163% of control, respectively) and LH (144, 182, and 221% of control, respectively) at concentrations of 75, 150, and 300 mg/kg/day. Serum PRL concentrations were significantly increased at 150 and 300 mg/kg/day (141 and 135% of control, respectively). Serum T, E2, and DHT concentrations were not affected by VCZ treatment at the dose levels tested. Thyroid Weight, Thyroid Hormone Concentrations, and Thyroid Histopathology (Table 4). Measures of thyroid function were included in order to evaluate the intact male assay for its ability to detect compounds that alter thyroid hormone homeostasis. CPA administration caused a statistically significant increase in relative thyroid weight at all dose levels (125, 125, 150, and 125% of control at dosages of 1, 10, 50, and 100 mg/kg/day, respectively). Serum TSH concentrations were significantly increased at 100 mg/kg/day (119% of control). Serum T 4 concentrations were significantly decreased at 50 and 100 mg/kg/day (72 and 63% of control, respectively). Serum T 3 concentrations were not affected by CPA treatment at the dose levels tested, and there were no compound-related microscopic changes in the thyroid gland. DDE administration caused a statistically significant increase in relative thyroid weight at all dose levels (150, 125, 150, and 125% of control at 50, 100, 200, and 300 mg/kg/day, respectively). Serum T 3 concentrations were significantly decreased at 100, 200, and 300 mg/kg/day (89, 89, and 86% of control, respectively). Serum T 4 concentrations were decreased in a dose-dependent manner and were significantly decreased at all dosages (82, 67, 56, and 51% of control at 50, 100, 200, and 300 mg/kg/day, respectively). Serum TSH concentrations were not affected by DDE treatment at the dose levels tested, and there were no compound-related microscopic changes in the thyroid gland. DBP administration caused a dose-dependent and statistically significant decrease in serum T 3 concentrations at all dose levels (83, 70, 52, and 45% of control at 250, 500, 750, and 1000 mg/kg/day, respectively). Serum T 4 concentrations were

98

O’CONNOR, FRAME, AND LADICS

TABLE 3 Intact Male Assay: Reproductive Hormone Concentrations Dosage Cyproterone acetate 0 1 10 50 100 p,p⬘-DDE 0 50 100 200 300 Di-n-butyl phthalate 0 250 500 750 1000 Flutamide 0 5 20 50 100 Linuron 0 25 c 50 a 100 a 150 Vinclozolin 0 10 75 150 300

T

E2

3.0 ⫾ 0.4 4.3 ⫾ 0.5 4.9 ⫾ 0.5* 5.0 ⫾ 0.7* 4.7 ⫾ 0.6*

8.6 ⫾ 0.8 16.4 ⫾ 0.6* 22.5 ⫾ 1.7* 24.9 ⫾ 1.4* 24.7 ⫾ 1.6*

2.9 ⫾ 0.4 2.9 ⫾ 0.5 2.3 ⫾ 0.4 b 2.5 ⫾ 0.6 2.6 ⫾ 0.4

DHT

PRL

FSH

LH

176.1 ⫾ 27.8 188.8 ⫾ 28.8 168.7 ⫾ 20.1 163.7 ⫾ 36.8 166.8 ⫾ 32.6

13.4 ⫾ 1.6 14.6 ⫾ 2.9 8.2 ⫾ 1.3* 3.8 ⫾ 0.4* 3.5 ⫾ 0.5*

11.7 ⫾ 1.1 11.5 ⫾ 0.6 11.6 ⫾ 0.5 10.5 ⫾ 0.7 9.2 ⫾ 0.8*

3.4 ⫾ 0.3 3.3 ⫾ 0.3 3.1 ⫾ 0.4 3.1 ⫾ 0.3 3.0 ⫾ 0.4

12.7 ⫾ 1.4 a 15.3 ⫾ 1.2 a 14.9 ⫾ 1.1 15.1 ⫾ 1.5 18.1 ⫾ 1.2*

177.4 ⫾ 19.4 144.4 ⫾ 13.8 112.1 ⫾ 11.4* 137.7 ⫾ 14.9* 114.8 ⫾ 8.2 a,*

14.0 ⫾ 2.7 11.2 ⫾ 2.5 11.7 ⫾ 2.0 14.9 ⫾ 3.2 14.6 ⫾ 3.1

5.8 ⫾ 0.4 a 5.9 ⫾ 0.3 5.9 ⫾ 0.4 5.2 ⫾ 0.3 5.3 ⫾ 0.2

3.2 ⫾ 0.2 2.9 ⫾ 0.2 3.4 ⫾ 0.2 2.8 ⫾ 0.2 2.8 ⫾ 0.1

3.7 ⫾ 0.4 3.1 ⫾ 0.4 2.5 ⫾ 0.4* 2.7 ⫾ 0.6* 2.0 ⫾ 0.3*

12.2 ⫾ 1.4 c 4.1 ⫾ 0.6 c,* 1.6 ⫾ 0.3* 1.2 ⫾ 0.3* 2.1 ⫾ 0.2* ,a

143.6 ⫾ 15.2 135.2 ⫾ 18.1 141.1 ⫾ 19.4 154.3 ⫾ 24.2 98.3 ⫾ 10.5

23.7 ⫾ 4.3 18.8 ⫾ 3.2 14.4 ⫾ 2.0 14.9 ⫾ 2.2 15.3 ⫾ 2.9*

12.7 ⫾ 0.6 13.7 ⫾ 1.0 13.7 ⫾ 0.4 13.7 ⫾ 0.7 15.0 ⫾ 0.6*

3.4 ⫾ 0.5 6.9 ⫾ 0.7* 10.4 ⫾ 1.2* 11.3 ⫾ 1.5* 10.6 ⫾ 1.2*

7.4 ⫾ 1.1 14.4 ⫾ 0.9* 16.1 ⫾ 1.1* 23.3 ⫾ 1.2* 30.1 ⫾ 2.6*

127.7 ⫾ 23.1 275.9 ⫾ 35.0 c,* 477.1 ⫾ 61.4 a,* 503.0 ⫾ 70.8* 416.4 ⫾ 74.7 c,*

10.6 ⫾ 1.5 12.0 ⫾ 2.0 12.1 ⫾ 1.4 11.3 ⫾ 1.2 9.1 ⫾ 1.2

12.0 ⫾ 0.7 15.7 ⫾ 0.5* 22.4 ⫾ 1.3* 25.3 ⫾ 1.1* 23.2 ⫾ 1.3*

3.7 ⫾ 0.3 5.5 ⫾ 0.3* 7.8 ⫾ 0.4* 10.0 ⫾ 0.6* 9.4 ⫾ 0.6*

4.5 ⫾ 0.5 4.7 ⫾ 0.6 5.0 ⫾ 0.5 4.2 ⫾ 1.1 2.7 ⫾ 0.5*

9.6 ⫾ 0.7 10.7 ⫾ 1.1 14.4 ⫾ 1.0* 15.8 ⫾ 1.6* 17.3 ⫾ 1.6*

248.6 ⫾ 33.5 230.8 ⫾ 28.7 234.8 ⫾ 40.8 173.7 ⫾ 44.2* 91.4 ⫾ 14.4*

10.1 ⫾ 1.3 10.3 ⫾ 2.3 10.2 ⫾ 2.3 10.7 ⫾ 2.1 4.8 ⫾ 0.6*

13.6 ⫾ 0.4 15.4 ⫾ 1.3 16.4 ⫾ 0.9 14.3 ⫾ 0.6 15.2 ⫾ 0.9

3.9 ⫾ 0.3 4.0 ⫾ 0.4 4.1 ⫾ 0.3 3.5 ⫾ 0.3 3.1 ⫾ 0.4*

2.5 ⫾ 0.4 3.1 ⫾ 0.5 2.7 ⫾ 0.4 2.9 ⫾ 0.5 3.7 ⫾ 0.5

7.5 ⫾ 1.0 9.1 ⫾ 0.6 7.9 ⫾ 0.7 7.3 ⫾ 1.0 7.8 ⫾ 0.9

116.5 ⫾ 19.1 153.7 ⫾ 24.2 125.8 ⫾ 15.9 111.2 ⫾ 17.9 140.9 ⫾ 21.6

7.8 ⫾ 0.7 10.6 ⫾ 1.7 9.8 ⫾ 1.2 11.0 ⫾ 1.3* 10.5 ⫾ 0.8*

14.4 ⫾ 0.7 15.0 ⫾ 1.0 17.7 ⫾ 1.0* 24.0 ⫾ 1.4* 23.4 ⫾ 1.4*

3.4 ⫾ 0.2 3.7 ⫾ 0.3 4.9 ⫾ 0.5* 6.2 ⫾ 0.5* 7.5 ⫾ 0.7*

2.4 ⫾ 0.2 2.6 ⫾ 0.2 2.9 ⫾ 0.2* 3.0 ⫾ 0.3* ,a 3.0 ⫾ 0.2*

Note. Values are mean ⫾ SE; n ⫽ 15 unless otherwise noted. Dosage is given in mg/kg/day. T, testosterone (ng/ml); E2, estradiol (pg/ml); DHT, dihydrotestosterone (pg/ml); PRL, prolactin (ng/ml); FSH, follicle stimulating hormone (ng/ml); LH, luteinizing hormone (ng/ml). a n ⫽ 14. b n ⫽ 12. c n ⫽ 13. *Significantly different (p ⬍ 0.05) from control by Jonckheere’s test for trend.

decreased in a dose-dependent manner and were significantly decreased at 500, 750, and 1000 mg/kg/day (50, 29, and 24% of control, respectively). Relative thyroid weights and serum TSH concentrations were not affected by DBP treatment at the dose levels tested, and there were no compound-related microscopic changes in the thyroid gland. FLUT administration caused a statistically significant decrease in serum T 4 concentrations at 100 mg/kg/day (66% of control). Relative thyroid weights and serum TSH and T 3 concentrations were not affected by FLUT treatment at the dose levels tested. There were no compound-related microscopic changes in the thyroid gland.

LIN administration caused a statistically significant decrease in serum T 3 concentrations at 50, 100, and 150 mg/kg/day (84, 84, and 78% of control, respectively). Serum T 4 concentrations were decreased in a dose-dependent manner and were significantly decreased at all dosages (72, 57, 43, and 30% of control at 25, 50, 100, and 150 mg/kg/day, respectively). Relative thyroid weights and serum TSH concentrations were not affected by LIN treatment at the dose levels tested, and there were no compound-related microscopic changes in the thyroid gland. VCZ administration caused a statistically significant increase in relative thyroid weight at 75 and 300 mg/kg/day (125

99

SCREENING FOR ANTIANDROGENS

TABLE 4 Intact Male Assay: Thyroid Parameters Dosage Cyproterone acetate 0 1 10 50 100 p,p⬘-DDE 0 50 100 200 300 Di-n-butyl phthalate 0 250 500 750 1000 Flutamide 0 5 20 50 100 Linuron 0 25 b 50 a 100 a 150 Vinclozolin 0 10 75 150 300

Thyroid weight

TSH

T3

T4

0.004 ⫾ 0.000 0.005 ⫾ 0.000* 0.005 ⫾ 0.000* 0.006 ⫾ 0.000* 0.005 ⫾ 0.000*

9.3 ⫾ 1.0 7.2 ⫾ 0.7 10.6 ⫾ 1.2 11.4 ⫾ 1.2 11.1 ⫾ 1.1#

84.2 ⫾ 3.3 93.9 ⫾ 3.6 101.5 ⫾ 4.3 101.2 ⫾ 3.6 89.9 ⫾ 4.0

4.3 ⫾ 0.2 3.6 ⫾ 0.2 3.9 ⫾ 0.2 3.1 ⫾ 0.1# 2.7 ⫾ 0.2#

0.004 ⫾ 0.000 0.006 ⫾ 0.000* 0.005 ⫾ 0.000* 0.006 ⫾ 0.000* 0.005 ⫾ 0.000*

15.8 ⫾ 1.4 17.1 ⫾ 1.4 18.6 ⫾ 1.9 17.9 ⫾ 1.4 18.6 ⫾ 1.5

85.2 ⫾ 1.8 78.2 ⫾ 3.1 75.7 ⫾ 3.1# 75.7 ⫾ 3.8# 72.9 ⫾ 2.5#

3.9 ⫾ 0.1 3.2 ⫾ 0.1# 2.6 ⫾ 0.1# 2.2 ⫾ 0.2# 2.0 ⫾ 0.1#

0.005 ⫾ 0.000 0.005 ⫾ 0.000 0.005 ⫾ 0.000 0.005 ⫾ 0.000 0.005 ⫾ 0.000

10.1 ⫾ 1.1 12.9 ⫾ 1.6 10.4 ⫾ 1.0 10.8 ⫾ 1.0 9.9 ⫾ 0.9

92.7 ⫾ 3.1 77.4 ⫾ 2.7# 65.2 ⫾ 2.9# 48.1 ⫾ 2.5# 41.7 ⫾ 2.5#

4.2 ⫾ 0.3 3.5 ⫾ 0.1 2.1 ⫾ 0.1# 1.2 ⫾ 0.2# a 1.0 ⫾ 0.1#

0.006 ⫾ 0.001 0.006 ⫾ 0.000 0.006 ⫾ 0.000 0.006 ⫾ 0.000 0.006 ⫾ 0.001

13.2 ⫾ 1.4 14.5 ⫾ 1.3 15.3 ⫾ 1.9 13.9 ⫾ 1.3 14.8 ⫾ 0.8

78.5 ⫾ 3.8 87.6 ⫾ 4.3 78.1 ⫾ 2.3 75.6 ⫾ 3.3 75.9 ⫾ 3.1

3.4 ⫾ 0.2 4.1 ⫾ 0.1 3.6 ⫾ 0.1 3.1 ⫾ 0.2 2.5 ⫾ 0.1#

0.005 ⫾ 0.000 0.005 ⫾ 0.000 0.006 ⫾ 0.000 0.006 ⫾ 0.000 0.006 ⫾ 0.000

14.0 ⫾ 1.2 10.8 ⫾ 1.1 12.4 ⫾ 1.2 14.5 ⫾ 1.4 11.8 ⫾ 0.8

84.1 ⫾ 3.6 80.9 ⫾ 3.5 70.8 ⫾ 2.9# 70.9 ⫾ 3.1# 65.7 ⫾ 4.3#

4.6 ⫾ 0.2 3.3 ⫾ 0.2# 2.6 ⫾ 0.2# 2.0 ⫾ 0.1# 1.4 ⫾ 0.2#

0.004 ⫾ 0.000 0.005 ⫾ 0.000 0.005 ⫾ 0.000* 0.005 ⫾ 0.001 0.006 ⫾ 0.000*

11.2 ⫾ 1.0 20.8 ⫾ 1.3 12.6 ⫾ 1.3 10.9 ⫾ 0.6 11.8 ⫾ 1.2

82.6 ⫾ 3.4 85.2 ⫾ 1.3 68.2 ⫾ 2.5# 63.0 ⫾ 2.2# 60.0 ⫾ 2.9#

4.1 ⫾ 0.1 3.5 ⫾ 0.2# 1.9 ⫾ 0.1# 1.2 ⫾ 0.1# 0.8 ⫾ 0.1#

Note. Values are mean ⫾ SE. The n ⫽ 15 unless otherwise noted. Dosage is given in mg/kg/day. Thyroid weight is given as % body weight. TSH, thyroid stimulating hormone (ng/ml); T 3 is given in ng/dl and T 4 in ␮g/dl. a n ⫽ 14. b n ⫽ 13. *Significantly different (p ⬍ 0.05) from control by Dunnett’s test. #Significantly different (p ⬍ 0.05) from control by Jonckheere’s test for trend.

and 150% of control, respectively). Serum T 3 concentrations were significantly decreased at 75, 150, and 300 mg/kg/day (83, 76, and 73% of control, respectively). Serum T 4 concentrations were decreased in a dose-dependent manner and were significantly decreased at all dosages (85, 46, 29, and 20% of control at 10, 75, 150, and 300 mg/kg/day, respectively). Serum TSH concentrations were not affected by VCZ treatment at the dose levels tested, and there were no compoundrelated microscopic changes in the thyroid gland. Immune system assessment (Table 5 and Fig. 1). DDE did not alter mean final body weights. Mean final body weights, however, were significantly decreased (93 and 91% of control,

respectively) in rats treated with 50 and 100 mg/kg/day FLUT. Neither DDE nor FLUT significantly altered spleen or thymus weights or spleen cell number. The primary humoral immune response to SRBC was not significantly altered by DDE or FLUT. As expected, the known immunosuppressant agent, CY, decreased the humoral immune response to SRBC (22–38% of control; data not shown). DISCUSSION

In this report, we examined six EACs with antiandrogenic activity in order to evaluate the sensitivity and specificity of the

100

O’CONNOR, FRAME, AND LADICS

TABLE 5 Immune System Assessment: Final Body and Organ Weights and Total Spleen Cell Number Final body weight Dosage (mg/kg/day) p,p⬘-DDE 0 50 100 200 300 Flutamide 0 5 20 50 100

Spleen

Thymus

g

% control

g

% BW

g

% BW

Total spleen cell number (⫻ 10 8)

417 ⫾ 18 417 ⫾ 22 423 ⫾ 39 424 ⫾ 40 408 ⫾ 25

100 100 101 102 98

0.816 ⫾ 0.121 0.814 ⫾ 0.079 0.818 ⫾ 0.112 0.814 ⫾ 0.113 0.780 ⫾ 0.090

0.195 ⫾ 0.025 0.196 ⫾ 0.022 0.193 ⫾ 0.019 0.192 ⫾ 0.021 0.191 ⫾ 0.016

0.506 ⫾ 0.115 0.531 ⫾ 0.075 0.594 ⫾ 0.135 0.565 ⫾ 0.173 0.520 ⫾ 0.090

0.121 ⫾ 0.026 0.127 ⫾ 0.017 0.139 ⫾ 0.023 0.134 ⫾ 0.042 0.128 ⫾ 0.026

10.09 ⫾ 3.63 10.33 ⫾ 2.78 10.79 ⫾ 1.91 10.99 ⫾ 2.76 10.74 ⫾ 1.59

429 ⫾ 26 422 ⫾ 21 406 ⫾ 29 398 ⫾ 31* 391 ⫾ 16*

100 98 95 93 91

0.786 ⫾ 0.101 0.770 ⫾ 0.103 0.733 ⫾ 0.106 0.792 ⫾ 0.138 0.718 ⫾ 0.099

0.185 ⫾ 0.032 0.182 ⫾ 0.020 0.181 ⫾ 0.025 0.198 ⫾ 0.029 0.184 ⫾ 0.022

0.465 ⫾ 0.107 0.515 ⫾ 0.128 0.509 ⫾ 0.201 0.427 ⫾ 0.099 0.418 ⫾ 0.082

0.109 ⫾ 0.030 0.122 ⫾ 0.028 0.124 ⫾ 0.045 0.106 ⫾ 0.019 0.107 ⫾ 0.020

9.04 ⫾ 3.16 9.69 ⫾ 2.71 8.87 ⫾ 2.70 8.63 ⫾ 2.79 8.32 ⫾ 1.63

Note. Mean ⫾ SE; n ⫽ 10. BW, body weight. *Significantly different (p ⬍ 0.05) from control by Dunnett’s Test.

15-day intact male assay for detecting EACs. The test substances that were evaluated included the antiandrogens CPA, DDE, DBP, FLUT, LIN, and VCZ. For each compound, the results were compared to the expected pattern of responses based on the known mechanism of action. Responses for DDE and FLUT were also compared to the responses that were obtained in previous studies where the compounds were administered via ip injection (O’Connor et al., 1998a, 1999a). In addition, an evaluation of immune system endpoints was conducted on a subset of animals dosed with either DDE or FLUT. Finally, the data from the current report were compared to data from the Hershberger and pubertal male assays in order to facilitate comparisons of the three screening assays for their ability to detect antiandrogens. The readers are also referred to the accompanying article (O’Connor et al., 2002), which presents data from four additional EACs (steroid biosynthesis inhibitors and thyroid modulators) that were evaluated via oral compound administration in the 15-day intact male assay. Detection of Antiandrogens In general, AR antagonists disrupt androgen homeostasis by competing for binding to the AR, resulting in displaced binding of endogenous androgens, effectively resulting in decreased intracellular androgenic stimulus and decreased recognition of androgens centrally. Ultimately, the pattern of the responses that would be expected for an AR antagonist would be decreased ASG weights and hormonal alterations (increased T, DHT, E2, LH, and FSH). Microscopic alterations of the testis may also be evident with some EACs; however, weak antiandrogens may not induce microscopic changes using a 15-day study duration (O’Connor et al., 1999a).

FIG. 1. Assessment of the primary IgM antibody response to SRBC following exposure to DDE or FLUT. Male rats (10/group) were exposed to a daily oral gavage dose of either 0, 50, 100, 200, or 300 mg/kg/day DDE or 0, 5, 20, 50, or 100 mg/kg/day FLUT for 15 days. Animals received SRBC by iv injection 6 days prior to termination for assessment of the humoral immune response. At termination, sera were obtained and analyzed for IgM antibody specific for SRBC by an ELISA as described in Materials and Methods. Results are reported as the log 2 of the SRBC-specific serum IgM titers.

SCREENING FOR ANTIANDROGENS

Of the six antiandrogens examined in the current report, four of them are AR antagonists (DDE, FLUT, LIN, and VCZ), one has mixed AR antagonist/PR agonist activity (CPA), and one is an antiandrogen that acts through a nonreceptor mediated mechanism (DBP). FLUT and VCZ showed the typical organ weight and hormonal pattern for an AR antagonist as described above. Both FLUT and VCZ caused Leydig cell hypertrophy and/or hyperplasia, and FLUT also caused spermatocyte degeneration and necrosis, all of which are characteristic effects of androgen deprivation within the testis (Russell et al., 1990a,b). The results for both compounds were consistent with previous reports (Gray et al., 1994; Neri, 1976; VanRavenzwaay, 1992). Furthermore, for FLUT the results obtained by gavage (current study) were similar in magnitude and scope to the effects that were reported by the authors when FLUT was administered via ip injection (O’Connor et al., 1998a). LIN, a weak AR antagonist (Cook et al., 1993; Lambright et al., 2000), produced the expected alterations on ASG weights, but did not produce the typical hormonal pattern for an AR antagonist. In contrast to a typical AR antagonist, LIN administration decreased serum T, DHT, and LH concentrations. These findings were unexpected since using a similar study design (i.e., 2-week treatment in intact adult male rats) with a lower number of animals per group (10 vs. 15 rats/group), Cook and coworkers (1993) illustrated that LIN administration induced the typical hormonal pattern associated with an AR antagonist. The reason for this discrepancy is unclear; however, it is likely that some of the variability associated with the hormonal changes could be attributed to the low intrinsic potency of LIN. Furthermore, in the report by Cook and coworkers (1993), the administered dose of LIN was greater than in the current study (150 vs. 200 mg/kg/day), although marked body weight effects were also observed in the study by Cook and coworkers. In the current study there was also evidence of spermatid retention, a manifestation of androgen deprivation within the testis (Russell et al., 1990a,b); this was not examined in the previous study by Cook and coworkers. Clearly, the data from the current study, which includes decreased ASG weights and microscopic alterations of the testis, combined with data from AR competition binding assays (Cook et al., 1993) (which would be included in any Tier I screen for EACs), would identify LIN as a weak AR antagonist. Based on those data, a Hershberger assay may be performed to confirm the weak AR antagonist activity of LIN. The results obtained with CPA are similar to data previously reported in the literature (Lakshman and Isaac, 1973; Neri, 1976; Neumann, 1982; Neumann and Topert, 1986), and are consistent for a compound with mixed AR antagonist/PR agonist activity. Similar to the effects observed with four of the six antiandrogens in the current report, ASG weights were decreased by CPA treatment, an effect consistent with an AR antagonist mechanism. However, this pattern of decreased ASG weights is also consistent with PR agonist activity. For example, in a previous report by the authors (O’Connor et al.,

101

2000b), administration of progesterone via oral gavage under the same study conditions also resulted in decreased ASG weights. Therefore, it is likely that the decreased ASG weights were the result of both the AR antagonist activity and PR agonist activity of CPA. The hormonal pattern for CPA had some similarities to an AR antagonist, as well as some similarities to a PR agonist. As stated previously, AR antagonists with no other intrinsic hormonal activity (e.g., FLUT) typically increase serum concentrations of LH to stimulate the Leydig cells to produce more T (and subsequently the metabolic products DHT and E2) as a mechanism to compensate for the decreased androgenic stimulus. CPA caused this pattern in serum T and E2, but not in LH. In contrast, PR agonists will decrease serum concentrations of LH and FSH as a consequence of the normal physiological feedback inhibition of gonadotropins by progestins at the anterior pituitary, and ultimately decrease production of T from the testis (Ascoli and Segaloff, 1990; Hsueh and Billig, 1995). CPA decreased serum LH and FSH concentrations. Under the appropriate conditions (i.e., sufficient dose and time of dosing), PR agonists will result in Leydig cell atrophy due to decreased steroidogenesis, an effect that has been observed for progesterone (O’Connor et al., 2000b) and other PR agonists (Chaudhary et al., 1990). In the current study, CPA caused microscopic alterations of the testis characterized by spermatocyte degeneration and necrosis, a finding consistent with androgen deprivation (Russell et al., 1990a,b). Overall, the endpoint profile for CPA was consistent with its mixed AR antagonist/PR agonist activity. DBP as an antiandrogen is unique since neither it nor its primary metabolite (monobutyl phthalate) interact with the AR in vitro (Foster et al., 2001). The mechanism for the DBP effects on reproduction and development is unknown, although many of the effects that DBP causes are consistent with an antiandrogen-like mechanism (Foster et al., 2001; Mylchreest et al., 1998, 1999, 2000; Srivastava et al., 1990a,b). A recent report by Shultz and coworkers (2001) illustrates that some of the antiandrogenic effects of DBP may be the result of altered gene expression patterns in the testis. In the report by Schultz and coworkers, gene expression in the fetal rat testis was evaluated after in utero exposure to DBP. They observed that one of the effects of DBP appears to be a down-regulation of the steroidogenic enzymes within the testis, a finding that also correlated with decreased concentrations of fetal testicular T (Mylchreest et al., 2002; Shultz et al., 2001). These data are consistent with the effects that have been observed on reproduction and development (Foster et al., 2001; Mylchreest et al., 1998, 1999, 2000; Srivastava et al., 1990a,b), as well as the results obtained in the current study using the 15-day intact male assay. In the current report, effects of DBP administration included decreased T, E2, and DHT; and increased serum LH and FSH concentrations. These results are also similar to those observed for ketoconazole, a testosterone biosynthesis inhibitor (see accompanying article, O’Connor et al., 2002). Although ketoconazole administration also resulted in decreased

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ASG weights, effects on ASG weights were not observed with DBP, reflecting the low potency of DBP or possibly the different mechanism(s) of action. In the intact male assay, DBP also caused microscopic alterations of the testis that included slight, multifocal degeneration in seminiferous tubules, as well as evidence of germ cell loss in the epididymides. These results are similar to previous results for DBP in immature as well as mature rats (Mylchreest et al., 1998, 1999, 2000; Srivastava et al., 1990a,b). Overall, the data that were obtained in the intact male assay are consistent with the proposed mechanism(s) of action for DBP (Foster et al., 2001; Mylchreest et al., 1998, 1999, 2000; Shultz et al., 2001; Srivastava et al., 1990a,b). Clearly the intact male assay identified DBP as an EAC that targets the testis and causes decreased T synthesis and subsequent hormonal sequellae consistent with altered androgen homeostasis. DDE, a weak AR antagonist (Kelce et al., 1995), has been previously examined in the intact male assay via the ip route of administration (O’Connor et al., 1999a). In that report, DDE was examined in Sprague Dawley and Long-Evans (LE) rats in order to examine possible strain-sensitivity differences to DDE. When administered by ip injection, DDE was not identified as an AR antagonist using Sprague-Dawley rats; however, using LE rats DDE was identified as an AR antagonist (O’Connor et al., 1999a). These data demonstrate that there are strain-sensitivity differences to DDE exposure using the 15day intact male assay, and they are consistent with strain differences in DDE metabolism (O’Connor et al., 1999a; You et al., 1998). The data for the current study where DDE was administered to Sprague-Dawley rats via oral gavage were similar to those observed when DDE was administered to Sprague-Dawley rats via ip injection; no definitive AR antagonist effects were observed. Serum estradiol concentrations were increased, a finding consistent with the data for DDE by the ip route of administration (O’Connor et al., 1999a). In addition, serum DHT concentrations were decreased, a response similar to that observed for DBP and LIN. However, overall the hormonal alterations for DDE were not consistent with the pattern typically observed with known antiandrogens. Therefore, DDE was considered a false-negative response in the intact male assay. This example (i.e., DDE) illustrates that many factors such as strain selection or even environmental conditions can contribute to the success or failure of a specific assay for detecting certain weak EACs. Clearly, all data should be considered when selecting appropriate screening assays. Furthermore, no one screening model will be 100% predictive or guaranteed to provide no false-positive or false-negative responses. However, in this instance, the intact male assay would have identified DDE as a potential thyroid toxicant based on the thyroid parameters, and as a potential AR antagonist based on AR binding data (O’Connor et al., 1999a), further illustrating the need for an integrated screening approach consisting of both in vitro and in vivo endpoints.

Evaluation of Thyroid Parameters In the 15-day intact male assay, thyroid parameters (thyroid weight, hormone analyses, and microscopic evaluation of the thyroid gland) were evaluated for their utility in detecting thyroid modulators. The expectation was that CPA and DDE would alter thyroid function since they have been shown to alter thyroid hormone homeostasis in previous studies (Bosland et al., 1992; Fitzhugh and Nelson, 1947; O’Connor et al., 1999a; Schreiber et al., 1971a,b), while DBP, FLUT, LIN, and VCZ would not alter thyroid function since they were negative for thyroid lesions in long-term rodent studies (U.S. EPA, 2002; Physician’s Desk Reference, 1999). As expected, DDE and CPA exhibited a thyroid endpoint profile that was consistent with a potential thyroid modulator (i.e., increased relative thyroid weight; increased TSH, decreased T 3/T 4), although neither compound caused microscopic alterations of the thyroid gland. For DDE, the alterations on thyroid hormone homeostasis are a result of enhanced clearance of T 4 due to an induction of hepatic UDP-glucuronyltransferase (Saito et al., 1991). The mechanism(s) for the alterations in thyroid parameters due to CPA administration are less well defined, although several publications implicate CPA in altered thyroid hormone homeostasis (Bosland et al., 1992; Schreiber et al., 1971a,b), and it is possible that the thyroid hormone alterations may be due to liver enzyme induction based on the increases in relative liver weight. These data support the hypothesis and are consistent with previous reports that CPA and DDE may be weak thyroid modulators. Thyroid hormone concentrations were also affected by DBP, FLUT, LIN, and VCZ, although only VCZ also affected relative thyroid weight. It is hypothesized that the decreases in T 3 and/or T 4 observed with all four EACs may be due to liver enzyme induction (i.e., increased hepatic clearance) based on the increases in relative liver weights. These data are also consistent with several other EACs that have been tested in the 15-day intact male assay (O’Connor et al., 1998a,b, O’Connor et al., 1999b; 2000a,b). Based on these data for thyroid parameters, and as previously discussed by the authors (O’Connor et al., 1999b), data interpretation based solely on changes in thyroid hormone levels is not sufficient for identifying thyroid modulators. For example, of the 28 compounds that we have evaluated in the intact male assay, 27 of the compounds produced a statistically significant change in at least one of the thyroid hormones. Hence, many compounds can transiently alter thyroid hormone homeostasis in rodents without resulting in long-term (i.e., adverse) thyroid effects. Therefore, one must be particularly cognizant of the fact that thyroid hormone concentrations are easily perturbed by a wide variety of chemicals, and a large proportion of these will not be thyroid toxicants in long-term studies. A more comprehensive approach such as the one taken by the 15-day intact male assay, including thyroid weight, thyroid hormone analyses, and microscopic evaluation of the thyroid gland appears prudent when

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SCREENING FOR ANTIANDROGENS

TABLE 6 Summary of Results from the Intact Male Assay Final body and organ weights

Microscopic changes

Serum hormone concentrations

Compound a

Body

Testes

Epididymides

ASG

Testes

Epididymides

T

DHT

E2

FSH

LH

PRL

Cyproterone acetate (100) p,p⬘-DDE (300) Di-n-butyl phthalate (1000) Flutamide (100) Linuron (150) Vinclozolin (300)

2 — — 2 2 —

— — — 2 — —

2 — — 2 2 2

2 — — 2 2 2

Yes — Yes Yes Yes Yes

— — Yes Yes — NE

1 — 2 1 2 —

— 2 — 1 2 —

1 2 2 1 1 —

2 — 1 1 — 1

— — 1 1 2 1

2 — 2 — 2 1

Note. ASG, accessory sex glands; T, testosterone; E2, estradiol; PRL, prolactin. The endpoint responses are noted by the following symbols: 1, increased; 2, decreased; or —, not affected. NE, not evaluated. a Highest dose level evaluated is shown in parentheses.

screening for compounds that target the thyroid gland. Thyroid modulators would be identified based on a weight-of-evidence approach using all the thyroid endpoints included in the 15-day intact male assay. Based on the responses seen with ⬎ 25 EACs evaluated to date (O’Connor et al., 1998a,b, 1999a,b, 2000a,b, 2001, 2002), thyroid toxicants should produce the following pattern of responses: a hormonal pattern of increased TSH and decreased T 3 and/or T 4 coupled with increased thyroid gland weight and/or histopathologic changes (colloid depletion and/or follicular cell hypertropy/hyperplasia). These changes may not always be statistically significant, and all thyroid parameters may not always be altered. In contrast, the absence of this characteristic pattern of changes suggests that the evaluated compound is not a thyroid toxicant even if statistically significant hormonal changes are observed, particularly when decreases in T 3 and/or T 4 are accompanied by increases in relative liver weights. Comparison of the Intact Male Assay and Other Screening Assays The results from the current report were also used to help facilitate a comparison of several of the assays that differ between the three proposed EDSTAC screening batteries (Table 1). Of the six antiandrogens examined in the current report, 5 have been evaluated in the Hershberger assay or the pubertal male assay. The Hershberger assay has been widely used in the pharmaceutical industry for years for identification of antiandrogens (Hershberger et al., 1953). The pubertal male assay is an apical test that relies primarily on evaluation of age at pubertal onset and organs weights for identifying EACs (EDSTAC, 1998). Limited hormonal assessment and histopathological evaluation are also included to enhance the sensitivity of the assay. The primary marker for pubertal onset is the age at preputial separation (PPS; androgen-dependent; Stoker et al., 2000). A comparison of the data for the antiandrogens that have been evaluated by the intact male assay, Hershberger assay, and pubertal male assay were surprisingly consistent (i.e., the

overall ability of each assay to detect them; Tables 6, 7, and 8). Of the four antiandrogens evaluated in all three assays, FLUT and VCZ, the most potent AR antagonists evaluated in the current report, were consistently identified as compounds that alter androgen status. Androgen-dependent organ weights were unequivocally decreased in all three assays. In the intact male assay, the hormonal pattern and histopathology assessment were useful in identifying the mode-of-action. Unfortunately, PPS data and the optional endpoints in the pubertal male assay were not evaluated. In addition, since only the intact male assay has evaluated these EACs under a dose-response study design, it is difficult to compare assay sensitivity. CPA, which was evaluated in the intact male and pubertal male assays, was also unequivocally identified in both assays. Similar to the data for FLUT and VCZ, the hormonal and histopathology assessments included in the intact male assay facilitated identification of the mode-of-action of CPA. LIN, which was evaluated in the intact male and the Hershberger assays, was definitively identified as an EAC that alters androgen status in both assays based on alterations in organ weights and/or serum hormones. Although the mode of action was not elucidated, LIN-induced alterations in serum hormones were observed at dosages of ⱖ 25 mg/kg/day; only a single dose (100 mg/kg/day) was evaluated in the Hershberger assay. The data for DDE and DBP were more difficult to interpret. As previously discussed, DDE was not identified as an EAC in the intact male assay. In the Hershberger and pubertal male assays, which both had multiple experiments that have evaluated DDE, data were inconsistent. In some experiments, DDE produced the expected pattern of effects for an antiandrogen, while in other experiments DDE caused no significant changes. For DBP, the intact male assay positively identified it as an EAC that altered androgen status based on the serum hormone profile coupled with the microscopic alterations of the testis and epididymis. The inconsistencies between experiments for DDE and DBP in the Hershberger and pubertal male assays are most likely the result of differences in study design parameters

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O’CONNOR, FRAME, AND LADICS

TABLE 7 Summary of Results from Hershberger Assays Conducted in Other Laboratories Final body and organ weights Compound a

No. experiments

Body

Seminal vesicles

Prostate

Ventral prostate

LABC

Cowper’s glands

p,p⬘-DDE (300) b,c,d,e,f Di-n-butyl phthalate (1000) c Flutamide (100) b,c,d,e,g,h Linuron (100) i Vinclozolin (100) c,i

11 11 22 1 3

— — — — —

2/— 2/— 2 2 2

2/— 2/— 2 NE 2

2/— 2/— 2 2 2

2/— 2/— 2 2 2

2/— 2/— 2 NE 2

Note. Includes assays using similar study designs to the Hershberger assay recommended by EDSTAC. LABC, levator ani plus bulbocavernosus. The endpoint responses are noted by the following symbols: 1, increased; 2, decreased; —, not affected; or 2/—, variable. NE ⫽ not evaluated. This table is a overall summary of all of the data available and represents what was most often observed with a particular test substance, and does not indicate that all experiments had the specified response and/or had all endpoints evaluated. a Highest dose level evaluated is shown in parentheses. b O’Connor et al., 1999a. c Ashby and Lefevre, 2000b. d Yamada et al., 2001. e Nellemann et al., 2001. f Sunami et al., 2000. g Yamada et al., 2000. h Yamasaki et al., 2001. i Lambright et al., 2000.

such as the age of the rats, the dose of testosterone administered, and the duration of dosing. Clearly, experimental design plays a critical role in the ability of a particular screening assay to detect EACs. Assessment of Immune Function Exposure of animals to the organochlorine pesticide 1,1,1trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), the parent of DDE, has been reported to alter both humoral and cell-medi-

ated immune responses (Barnett and Rogers, 1994). In the present study, however, DDE did not significantly alter the primary humoral immune response to SRBC nor alter spleen and thymus weights or spleen cell number. In contrast to our findings, Banerjee and coworkers (1996) reported that 200 ppm DDE administered in the diet of rats for six weeks significantly decreased the OVA specific-IgG antibody response. The differences observed between our results and those of Banerjee and coworkers (1996) may be due to differences in the ages,

TABLE 8 Summary of Results from Pubertal Male Assays Conducted in Other Laboratories Final body and organ weights Compound a

No. experiments

Body

Testes

Epididymides

Seminal vesicles

Prostate

Age at PPS

Cyproterone acetate (25) b,c p,p⬘-DDE (150) c,d Di-n-butyl phthalate (500) c Flutamide (25) c,e,f Vinclozolin (100) b,c

3 5 3 3 3

2 — — — —

2 — 2/— — —

2 2/— 2/— 2 2

2 2/— 2/— 2 2

2 — — 2 —

NE — — NE NE

Note. PPS, preputial separation.The endpoint responses are noted by the following symbols: 1, increased; 2, decreased; —, not affected; or 2/—, variable. NE ⫽ not evaluated. Includes assays using similar study designs to the pubertal male assay recommended by EDSTAC with at least 14 days of compound administration. a Highest dose level evaluated is shown in parentheses. b Ashby and Lefevre, 1997. c Ashby and Lefevre, 2000a. d Yamada et al., 2001. e Miyata et al., 2001. f Yamada et al., 2000.

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TABLE 9 15-Day Intact Adult Male Assay: Historical Control Data from 28 Studies Using 10-Week-Old (ⴞ 2 weeks) Sprague-Dawley Rats Study date

BW

Liver

Testes

ASG

Epididymides

Prostate

Seminal vesicles

T

DHT

E2

PRL

LH

FSH

TSH

T3

T4

November 1996 January 1997 March 1997 May 1997 July 1997 October 1997 November 1997 January 1998 February 1998 March 1998 May 1998 June 1998 August 1998 October 1998 October 1998 November 1998 February 1999 May 1999 July 1999 September 1999 October 1999 August 2000 September 2000 October 2000 September 2001 October 2001 November 2001 December 2001 Mean STDEV % CV

386 407 402 417 397 404 394 409 411 388 413 427 421 414 380 427 416 431 427 440 420 410 422 415 408 424 422 428 413 14 4

3.7 3.9 3.9 4.1 3.9 3.9 3.9 4.0 3.8 3.9 3.9 3.8 4.0 3.9 3.9 4.1 3.9 3.8 3.8 3.9 4.1 3.9 3.9 3.8 3.9 4.0 4.2 4.2 3.9 0.1 3

3.2 3.1 3.1 3.2 3.1 3.1 3.0 3.1 3.0 3.1 3.3 3.3 3.2 3.3 3.3 3.3 3.3 3.2 3.3 3.4 3.4 3.1 3.2 3.3 3.3 3.3 3.3 3.3 3.2 0.1 3

0.619 0.551 0.603 0.553 0.570 0.609 0.618 0.599 0.630 0.583 0.596 0.525 0.556 0.552 0.675 0.604 0.594 0.604 0.588 0.578 0.602 0.562 0.587 0.596 0.597 0.559 0.532 0.573 0.586 0.032 5

1.22 1.12 1.27 1.19 1.19 1.20 1.16 1.16 1.21 1.15 1.22 1.26 1.21 1.14 1.21 1.27 1.18 1.19 1.26 1.22 1.28 1.12 1.16 1.16 1.16 1.15 1.02 1.20 1.19 0.06 5

0.197 0.182 0.232 0.222 0.228 0.203 0.213 0.208 0.203 0.214 0.189 0.198 0.173 0.149 0.224 0.191 0.174 0.182 0.19 0.166 0.172 0.172 0.172 0.157 0.153 0.158 0.143 0.169 0.187 0.025 13

0.409 0.348 0.339 0.294 0.334 0.389 0.392 0.379 0.411 0.352 0.393 0.319 0.369 0.396 0.433 0.393 0.415 0.419 0.39 0.41 0.432 0.387 0.412 0.437 0.443 0.437 0.392 0.407 0.389 0.037 9

3.3 2.4 3.6 2.9 2.7 4.1 3.0 2.5 3.2 3.6 3.0 3.0 3.3 3.5 3.7 1.9 2.3 2.4 3.4 2.6 2.9 2.5 3.0 4.4 4.5 3.0 3.7 3.4 3.1 0.6 20

115.9 103.1 155.7 123.7 87.7 160.3 77.6 162.5 57.2 118.6 104.6 51.5 191.4 162.3 147.4 93.5 103.6 152.8 127.7 151.0 177.4 116.5 176.1 202.2 248.6 162.9 143.6 178.5 137.6 44.8 33

20.9 16.4 8.9 8.7 10.2 11.5 11.6 6.2 11.3 9.7 5.1 4.4 10.8 11.1 11.4 6.2 5.3 10.8 7.4 13.9 12.7 7.5 8.6 8.4 9.6 9.7 12.2 5.6 9.9 3.6 36

8.0 6.2 10.1 6.4 7.4 11 7.8 7.7 14.4 9.3 12.1 10.3 12.8 17.9 11.2 11.8 12.5 11.8 10.6 7.2 14.0 7.8 13.4 7.8 10.1 20.3 23.7 18.9 11.5 4.4 38

4.4 3.3 4.4 3.5 4.2 4.5 3.3 3.7 4.6 3.3 4.9 2.9 4.5 4.4 3.9 3.8 4.7 3.1 3.7 2.9 3.2 3.4 3.4 2.9 3.9 3.7 2.4 3.2 14.4 3.1 22

20.3 84.7 a 20.7 16.3 21.4 13.1 13.7 15.9 16.1 16.3 16.1 14.5 16.1 13.1 13.8 12.5 12.4 13.7 12.0 12.7 5.8 14.4 11.7 12.8 13.6 12.7 12.7 14.9 3.7 0.7 18

15.1 24.1 15.2 15.4 15.6 18.1 18.2 14.5 18.3 18.6 18.2 13.4 14.6 19.0 15.6 18.1 15.4 14.3 13.2 14.4 15.8 11.2 9.3 11.8 14.0 14.8 10.1 13.5 15.4 3.1 20

60.9 59.2 70.9 82.1 83.9 59.0 67.7 78.9 73.4 71.5 74.1 55.3 62.1 80.7 58.9 71.9 76.3 85.3 78.5 93.0 85.2 82.6 84.2 87.9 84.1 73.3 92.7 67.3 75.0 10.7 14

3.5 2.9 2.7 3.5 3.2 2.9 2.8 2.7 3.1 4.1 3.8 2.9 2.7 4.3 3.6 3.3 3.3 5.1 3.4 3.8 3.9 4.1 4.3 4.4 4.6 4.4 4.2 3.3 3.6 0.7 18

Note. BW, body weight; ASG, accessory sex glands; T, testosterone; DHT, dihydrotestosterone; E2, estradiol; PRL, prolactin; LH, luteinizing hormone; FSH, follicle stimulating hormone; TSH, thyroid stimulating hormone. BW, testes, and epididymides weights are in g; liver, ASG, prostate, and seminal vesicles weights are expressed as % BW. T, PRL, LH, FSH, and TSH are given in ng/ml; T 3 is given in ng/dl; T 4 is given in ␮g/dl; DHT and E2 are in pg/ml. a Excluded from calculations due to high variability associated with one specific radioimmunoassay kit lot number.

strains, dosing regimens, and/or differences in the type, route, dose and timing of antigen administration. In addition, we evaluated the primary IgM response whereas Banerjee and coworkers (1996) evaluated the secondary IgG antibody response. Exposure to FLUT by gavage for 14 days did not significantly alter the primary humoral immune response to SRBC. These results are consistent with our previous findings in which the exposure of rats for 15 days to 1, 5, or 20 mg/kg/day FLUT by ip injection did not alter the primary antibody response to SRBC (Ladics et al., 1998). Similarly, the antibody response to SRBC of chickens treated i.m. from time of hatch to 27 days of age with FLUT was not significantly altered (Leitner et al., 1996). Conclusions Of the six antiandrogens evaluated, the 15-day intact male assay definitively identified CPA, DBP, FLUT, LIN, and VCZ,

but not the weak AR antagonist DDE. The data for FLUT and DDE are similar to the data that has been previously reported by the authors, where each compound was evaluated in the 15-day intact male assay using the ip route of compound administration (O’Connor et al., 1998a, 1999a). The data from the current report, in addition to the ⬎ 20 compounds that have already been examined using the 15-day intact male assay, support this assay as a viable alternative to the screening model recommended by EDSTAC (EDSTAC, 1998; O’Connor et al., in press). The authors envision the 15-day intact male assay as one component of a Tier I screening battery for identifying EACs (O’Connor et al., in press). One clear benefit to a screening battery utilizing the 15-day intact male assay over the apical approach recommended by EDSTAC is that it is a mode-of-action screening assay, with the ability not only to identify potential EACs, but to determine a specific mode of

106

O’CONNOR, FRAME, AND LADICS

action. This was demonstrated by the comparison of the results from the intact male, Hershberger, and pubertal male assays. Although the overall ability of each assay to identify the antiandrogens was very similar, the comprehensive hormonal assessment facilitated identification of the mode-of-action. In addition, serum hormone measurements are typically the most sensitive endpoint of the intact male assay. For all three screening assays, study designs must be consistent, and more EACs must be evaluated to more fully understand the strengths and limitations of each. These and previous data (Biegel et al., 1998; Ladics et al., 1998) with EACs also suggest that the reproductive and endocrine systems and not the immune system are the primary target organs of toxicity in young adult rats. However, it is premature to conclude that the immune system is not a primary target organ of toxicity. Further studies are needed to evaluate the effects of low-dose chronic exposures to EACs on humoral, cell-mediated, and innate immunity as well as the developing immune system. Regardless of the screening model that is chosen, there are study design issues that should be considered in assays used for EAC screening. For example, strain-sensitivity differences such as those observed for DDE can contribute to interlaboratory variability, as well as contribute to possible false-negative and false-positive responses. Use of the same rat strain that is used in multigeneration reproduction studies would facilitate assay comparisons. In addition, as screening assays are evaluated and validated, the use of the same strain of rat will facilitate development of historical control data such as that included for the 15-day intact male assay (Table 9). These data will also help to determine endpoint variability and/or stability over time, both of which can impact assay reliability when screening for EACs. Dose levels for evaluation of a compound should be selected in order to provide the highest sensitivity to endocrine-related changes without resulting in nonspecific effects on the endocrine system due to general toxicity, and dietary restriction experiments should be performed for each type of in vivo test to help differentiate between true compound-related effects on the selected endpoints from those that are a result of decreased body weight (O’Connor et al., 1999b, 2000b). As we continue to evaluate potential screening models, these are just a few of the issues that must be considered. The 15-day intact male assay appears to be a viable approach for screening for EACs, although it is not the most sensitive approach for identifying weak AR antagonists. For AR antagonists, the Hershberger assay is the most sensitive assay. However, all components, both in vitro and in vivo, must be considered in parallel when deciding which screening approaches are satisfactory. Clearly, this will be a challenge for the scientific community. Furthermore, only interassay comparisons with the same test substances in the context of a comprehensive screening battery will characterize the strengths and limitations of each of the potential screening assays and/or models, and ultimately determine the appropriate models that should be implemented. This report, as well as the accompanying article

(O’Connor et al., 2002) and the previously published data from the authors (O’Connor et al., 1998a,b, 1999a,b, 2000a,b, 2001) will provide a starting point for the critical evaluation of the 15-day intact male assay. ACKNOWLEDGMENTS We would like to acknowledge the invaluable advice of Dr. A. Michael Kaplan. We thank Vivian Thompson, Bryan Crossley, Suzanne Craven, Charlene Smith, Michele Egan, and Denise Janney for their technical support.

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