Key learnings from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 in vitro assays.

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17ß-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

Initial insights regarding the role of p53 in maintaining sperm DNA integrity following treatment of mice with ethylnitrosourea or cyclophosphamide.

If p53 is essential to eliminate damaged spermatogenic cells, then mutagen exposure in the absence of p53 would increase sperm containing damaged DNA. p53 knockout (-/-, NULL) and wild-type (+/+, WT) mice (five/group) were exposed to ethylnitrosourea (ENU) or cyclophosphamide (CP). In phase I, mice were exposed by gavage to 0 or 60 mg/kg/day ENU or CP for four days and examined on test day (TD) 4, and in phase II, mice were exposed to 0, 6, 20, or 60 mg/kg/day ENU or CP for four days and evaluated on TD 36 when exposed spermatocytes matured. In phase I, mutagens were not directly cytotoxic to mature sperm. In phase II, WT mice were more sensitive to decreases in reproductive organ weights, whereas both genotypes had decreased sperm counts. Testicular histology revealed similar CP responses, but genotype-specific ENU responses (WT mice had depletion of elongating spermatids; NULL mice had late-stage spermatocyte/early stage spermatid loss). Ethylnitrosourea increased DNA strand breaks in WT mice. Thus, mice responded similarly to CP, suggesting a primarily p53-independent response, whereas the ENU response differed by zygosity, suggesting a role for p53. As DNA damage increased at higher ENU doses, compensatory repair pathways may operate in NULL mice.

A comparison of in vitro and in vivo EDSTAC test battery results for detecting antiandrogenic activity.

The androgen receptor (AR) transactivation, binding, and Hershberger assays are being developed for large-scale screening of chemicals for endocrine activity. The goal of this study was to evaluate the correlation between in vitro and in vivo antiandrogenicity assays using a variety of compounds (p,p'-DDE, flutamide (FLUT), spironolactone, procymidone, RU486, methoxychlor (MXC), benzo(a)pyrene (BAP), and selected metabolites). For the AR transactivation assay, AR(+) LNCaP prostate carcinoma cells were transfected with an inducible luciferase reporter construct (pGudLuc7ARE) and exposed for 24 h to test materials (< or = 10 microM) in the presence and absence of 1 nM of the AR agonist R-1881. Each of these materials, including the hydroxlated metabolites of BAP and MXC, produced significant antiandrogenic activity in vitro as evidenced by their inhibition of the response to R-1881. Similarly, in vitro AR binding experiments using the recombinant ligand-binding domain (LBD) of the human AR and fluorescence polarization (FP) methodology yielded IC50s comparable to that of testosterone for RU486 and 9-OH-BAP. Other parent compounds and metabolites exhibited lesser binding affinity. In vivo antiandrogenic activity was evaluated with the Hershberger assay, wherein castrated male CD rats were dosed by gavage for 10 days with (mg/kg per day): MXC (10, 50, 100, and 200), BAP (1, 10, 50, and 100), RU486 (1, 5, 10, and 25), and FLUT (10) in the presence of 0.4 mg/kg per day (sc) of testosterone propionate (TP). Neither BAP nor MXC produced significant decreases in accessory sex tissue (AST) weights relative to TP control. However, 200 MXC resulted in a significant decrease in body weight and 100 BAP significantly increased absolute and relative liver weights. RU486 (25) produced significant decreases in ventral prostate, seminal vesicle, and Cowper's gland weights without affecting body weight. FLUT (10) decreased all AST weights measured. The antiandrogenic activities of the remaining materials (p,p'-DDE, spironolactone, and procymidone) have been demonstrated in previous Hershberger assays. These data indicate the importance of including in vivo results in assessing the endocrine activity of test materials and further stress the importance of a weight of evidence approach in assessing endocrine activity of test materials.