Mary Sue Marty

Evaluation of EPA's Tier 1 Endocrine Screening Battery and recommendations for improving the interpretation of screening results.

EPAs Endocrine Disruptor Screening Program (EDSP) was implemented in 2009-2010 with the issuance of test orders requiring manufacturers and registrants of 58 pesticide active ingredients and nine pesticide inert/high production volume chemicals to evaluate the potential of these chemicals to interact with the estrogen, androgen and thyroid hormone systems. The required endocrine screening will be conducted over the next 2-3years. Based on estimates of the impacted sectors, costs are at least

Endocrine Disruption: Historical Perspectives and its Impact on the Future of Toxicology Testing

750,000-

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

1,000,000 per substance if all of the Tier 1 assays must be conducted. The screening will entail evaluation of responses in EPAs Tier 1 Endocrine Screening Battery (EDSP ESB), consisting of 11 distinct in vitro and in vivo assays. We reviewed the details of each test method and describe the critical factors integral to the design and conduct of the EDSP ESB assays as well as the limitations related to specificity and sensitivity. We discuss challenges to evaluating each assay, identify significant shortcomings, and make recommendations to enhance interpretation of results. Factors that affect the length of time necessary to complete the EDSP ESB for any particular substance are presented, and based on the overall analysis, we recommend a sequence for running the EDSP ESB assays. It is imperative that a structured, systematic weight of evidence framework is promptly developed, subjected to peer review and adopted. This will help to ensure an objective analysis of the results of the required EDSP screening, consistent integration of results across the EDSP ESB assays, and consistent decision making as to whether subsequent testing for adverse effects is needed. Based upon the limitations of the current EPA EDSP ESB, we concur with the Agencys Scientific Advisory Panels recommendation that after the initial set of substances has been screened, the EDSP ESB should pause so that the results can be fully analyzed to determine the value of the existing assays. After this analysis, assays that are unnecessarily redundant or that lack endocrine specificity should be eliminated and if necessary, replaced by new or revised screens that are more mechanistically specific, rapid, reliable, and cost effective.

Life-Stage-, Sex-, and Dose-Dependent Dietary Toxicokinetics and Relationship to Toxicity of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Rats: Implications for Toxicity Test Dose Selection, Design, and Interpretation

The endocrine system is one of the body’s major homeostatic control systems whose aim is to maintain normal functions and development in the face of a constantly changing environment. Working in tandem with the nervous system, which mainly is responsible for rapid and immediate responses, the approximately 30 different glands comprising the endocrine system tend to act in a slower and more sustained manner to regulate processes as diverse as the female reproductive cycle, bone growth, cell proliferation, and psychosocial behavior. Multiple endocrine glands also work in concert with one another to form complex feedback loops, which tightly regulate critical physiological processes. Like all homeostatic control systems, the capacity to maintain physiological parameters within normal bounds is finite, and when this capacity is exceeded by chemical exposures, drugs, or environmental stressors, adverse consequences can ensue. In the mid-1990’s, concerns about the potential for environmental chemicals, drugs, and other stressors to alter endocrine physiology rapidly mounted and received a great deal of attention within the toxicology community as well as in the public media. These circumstances spurred a flurry of research on mechanisms of toxicity, new questions about how to deal with cumulative exposures to endocrine-active compounds (EACs) from man-made, dietary, and endogenous sources, and the creation of major chemical screening and testing programs focused on endocrine-mediated modes of toxic action. In this review, we will trace the path of knowledge regarding the effects of exogenous agents on the endocrine system, the events which gave rise to the explosion of interest in this topic in the mid-1990’s, and how it led to a fundamentally different paradigm in toxicity testing as well as a great deal of basic research on mechanisms of endocrine-mediated toxicity. We first cover the history of testing for endocrine-mediated toxicity, followed by coverage of key historical events in basic science, and then bring the two together to discuss implications of the science on testing both now and for the future.

Cholinesterase inhibition and toxicokinetics in immature and adult rats after acute or repeated exposures to chlorpyrifos or chlorpyrifos–oxon

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10–12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The “No Observed Adverse Effect Level” for systemic toxicity was 300 ppm in both males (16.6mg/kg/day) and females (20.6mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

Inter-laboratory control data for reproductive endpoints required in the OPPTS 870.3800/OECD 416 reproduction and fertility test

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥1200 ppm (63mg/kg/day) for P1 males and between 200 and 400 ppm (14–27mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21–35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.

Dynamic Changes in Lipids and Proteins of Maternal, Fetal, and Pup Blood and Milk during Perinatal Development in CD and Wistar Rats

The effect of age or dose regimen on cholinesterase inhibition (ChEI) from chlorpyrifos (CPF) or CPF-oxon (CPFO) was studied in Crl:CD(SD) rats. Rats were exposed to CPF by gavage in corn oil, rat milk (pups), or in the diet (adults) or to CPFO by gavage in corn oil. Blood CPF/CPFO levels were measured. With acute exposure, ChEI NOELs were 2 mg/kg CPF for brain and 0.5 mg/kg CPF for red blood cells (RBCs) in both age groups. In pups, ChEI and blood CPF levels were similar using either milk or corn oil vehicles. Compared to gavage, adults given dietary CPF (12 h exposure) had greater RBC ChEI, but lower brain ChEI at corresponding CPF doses, indicating an effect of dose rate. With repeated CPF exposures, ChEI NOELs were the same across ages (0.5 and 0.1 mg/kg/day for brain and RBCs, respectively). With CPFO dosing, the ChEI NOELs were 0.1 mg/kg (acute) and 0.01 mg/kg/day (repeated doses) for RBCs with no ChEI in brain at CPFO doses up to 0.5 (pup) or 10 mg/kg (adult) for acute dosing or 0.5 mg/kg/day for both ages with repeat dosing. Thus, there were no age-dependent differences in CPF ChEI via acute or repeated exposures. Pups had less ChEI than adults at comparable blood CPF levels. Oral CPFO resulted in substantial RBC ChEI, but no brain ChEI, indicating no CPFO systemic bioavailability to peripheral tissues.

Pronamide: Weight of evidence for potential estrogen, androgen or thyroid effects.

BACKGROUND The U.S. EPA revised the Reproduction and Fertility Effects Test Guideline (OPPTS 870.3800/OECD 416) in 1998, adding numerous endpoints in an effort to incorporate new methodologies, improve the sensitivity for detecting reproductive toxicants, and more efficiently utilize study animals. Many of these new endpoints have not been used in regulatory reproductive toxicology studies prior to their inclusion in the test guidelines; thus, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI) initiated the Reproductive Endpoints Project to examine the utility of these new endpoints. METHODS This report provides a retrospective analysis of 43 multi-generation studies (16 in Wistar rats, 27 in Sprague-Dawley rats) conducted according to the latest version of the test guidelines. It focuses on vehicle (negative) control values (means and ranges) for the various endpoints to examine inter-laboratory variability. RESULTS Based on the compiled data, the most variable endpoints across laboratories and their associated coefficients of variation (CV) for each generation were: percent abnormal sperm (166-205%), testicular spermatid concentration (126-147%), postimplantation loss (97-104%), primordial follicle counts (69%, only measured in P2 females), and epididymal sperm concentration (52-57%). Absolute and relative prostate and thymus weights, weanling uterine weights, and anogenital distance had CVs of 25-50%. Sources of variability included procedural differences between laboratories, inherent biological variability, and/or small sample sizes for some endpoints. CONCLUSIONS These inter-laboratory control data provide a means for laboratories to review their performance on reproductive toxicity measures, and provide perspective for interpreting their own control data and data from treated animals.

Does GLP enhance the quality of toxicological evidence for regulatory decisions

An understanding of the physiological factors that regulate perinatal dosimetry is essential to improve the ability of physiologically based (PB) pharmacokinetic (PK) models to predict chemical risks to children. However, the impact of changing maternal/offspring physiology on PK during gestation and lactation remains poorly understood. This research determined lipid and protein changes in blood, milk and amniotic fluid of CD and Wistar dams, fetuses and neonates to improve the precision of perinatal PBPK modeling. Samples were collected from time-mated CD dams, fetuses, and pups on gestation day (GD) 18 and 20 (sperm positive = GD 0) or lactation day 0 (day of birth), 1, 3, 5, 10, 15, and 20 (n > or = 5 per time point). Fewer time points were sampled in Wistar rats, which showed similar patterns to CDs. Relative to nonpregnant dams, maternal serum protein levels (albumin, total protein and globulin) each decreased by approximately 20% during late gestation, whereas maternal serum lipids (triglycerides, low density lipoproteins, and phospholipids) increased up to fourfold. These physiological changes can impact maternal PK of both protein-bound and lipophilic chemicals. During lactation, triglycerides in milk were greater than 100-fold higher than maternal serum, favoring the disposition of lipophilic chemicals into milk and potentially increasing neonatal rodent exposure during critical stages of postnatal development. Serum protein levels in pups were two- to threefold lower than adults at birth, which may increase the bioavailability of protein-bound compounds. These data will aid in the interpretation of perinatal toxicity studies and improve the accuracy of predictive perinatal PBPK models.

Weight-of-the-evidence evaluation of 2,4-D potential for interactions with the estrogen, androgen and thyroid pathways and steroidogenesis

Based on the exposure potential to humans and environment, pronamide was one of 52 chemicals on the first list evaluated under US EPAs Endocrine Disruptor Screening Program (EDSP). The purpose of EDSP is to screen chemicals for their potential to interact with estrogen-, androgen-, or thyroid-signaling pathways. A battery of 11 Tier 1 assays was completed for pronamide in accordance with EDSP test guidelines. In addition, Other Scientifically Relevant Information, which included existing data from regulatory guideline studies and published literature, was used in a weight-of-evidence (WoE) evaluation of potential endocrine activity. The WoE conclusion is that pronamide does not interact directly with estrogen, androgen, or thyroid receptors or post-receptor events. Across in vivo studies, the liver is consistently and reproducibly the target organ for pronamides effects. Pronamide activates hepatocytic nuclear receptors (including constitutive androstane receptor), induces hepatic enzymes, produces hepatocellular hypertrophy and increases liver weights. These changes are coupled with increased metabolic activity and a subsequent increased metabolism and/or clearance of both steroid and thyroid hormones. Thus, while pronamide alters some endocrine-sensitive endpoints in EDSP Tier 1 assays, effects on liver metabolism likely explain altered hormone levels and indirect endocrine changes.