Edward W. Carney

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

The rabbit as a model for reproductive and developmental toxicity studies

The rabbit has many advantages as a nonrodent and second model for assessing the effects of toxic agents on semen quality, fertility, developmental toxicity, and teratology. The male and female reproductive systems of the rabbit are described, and data on growth, sexual development and reproduction are compared with mice, rats, and humans. Techniques for semen collection and evaluation in the male, and artificial insemination, superovulation, embryo culture, and embryo transfer in the female are included as useful procedures in toxicity testing. Examples of the use of rabbits and experimental replication for toxicity testing are given. Special features of the visceral yolk sac and development of the chorioallantoic placenta of the rabbit are compared with rodents. The rabbit extraembryonic membranes more closely resemble the human than do the rodents, in some respects. The use of the rabbit in developmental toxicity and teratology studies is discussed.

Evaluation of Physiologically Based Models of Pregnancy and Lactation for Their Application in Children's Health Risk Assessments

In todays scientific and regulatory climates, an increased emphasis is placed on the potential health impacts for children exposed either in utero or by nursing to drugs of abuse, pharmaceuticals, and industrial or consumer chemicals. As a result, there is a renewed interest in the development and application of biologically based computational models that can be used to predict the dosimetry (or ultimately response) in a developing embryo, fetus, or newborn. However, fundamental differences between animal and human development can create many unique challenges. For example, unlike models designed for adults, biologically based models of pre- and postnatal development must deal with rapidly changing growth dynamics (maternal, embryonic, fetal, and neonatal), changes in the state of differentiation of developing tissues, uniquely expressed or uniquely functioning signal transduction or enzymatic pathways, and unusual routes of exposure (e.g., maternal-mediated placental transfer and lactation). In cases where these challenges are overcome or addressed, biological modeling will likely prove useful in assessments geared toward childrens health, given the contributions that this approach has already made in cancer and non-cancer human health risk assessments. Therefore, the purpose of this review is to critically evaluate the current state of the art in physiologically based pharmacokinetic (PBPK) and pharmacodynamic (PD) modeling of the developing embryo, fetus, or neonate and to recommend potential steps that could be taken to improve their use in childrens health risk assessments. The intent was not to recommend improvements to individual models per se, but to identify areas of research that could move the entire field forward. This analysis includes a brief summary of current risk assessment practices for developmental toxicity, with an overview of developmental biology as it relates to species-specific dosimetry. This summary should provide a general context for understanding the tension that exists in modeling between describing biological processes in exquisite detail vs. the simplifications that are necessary due to lack of data (or through a sensitivity analysis, determined to be of little impact) to develop individual PBPK or PD models. For each of the previously published models covered in this review, a description of the underlying assumptions and model structures as well as the data and methods used in model development and validation are highlighted. Although several of the models attempted to describe target tissues in the developing embryo, fetus, or neonate of laboratory animals, extrapolations to humans were largely limited to maternal blood or milk concentrations. Future areas of research therefore are recommended to extend the already significant progress that has been made in this field and perhaps address many of the technical, policy, and ethical issues surrounding various approaches for decreasing the uncertainty in extrapolating from animal models to human pregnancies or neonatal exposures.

Evaluation of Tier I screening approaches for detecting endocrine-active compounds (EACs).

In 1996, Congress passed legislation requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for endocrine-active compounds (EACs). In response, 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 or androgen receptors, steroid biosynthesis inhibitors, or their ability to alter thyroid function. For Tier I, the EDSTAC-recommended screening battery comprised eight different assays, but EDSTAC also proposed two alternative batteries that were deemed worthy of further evaluation. The challenge currently confronting EPA is to choose among the Tier I screening options and then to standardize protocols, validate the assays, and determine the criteria for judging a compound as positive or negative in the battery. The purpose of the current review is to: (1) provide an overview of the three EDSTAC options, (2) evaluate the data currently available for the individual assays of the three EDSTAC options and discuss the strengths and limitations of each, and (3) provide a final recommendation for a Tier I screen based on the experiences of the authors who have used all of the individual assays under consideration by EDSTAC. The goal of this report is not to provide an exhaustive historical review of each assay, but rather to summarize some of the more relevant data from available published reports as it relates to current proposed study designs for those particular assays. Based on the current data, a Tier I screening battery consisting of in vitro receptor binding assays, a 3-day uterotrophic assay, and a 15-day intact male assay are recommended as the preferred approach on which future validation efforts should be focused. This screening approach is a mode-of-action screen that will identify specific types of endocrine activity. Because it utilizes many endpoints from the same test animals (i.e., it integrates), it is the most cost-effective and efficient option in terms of animal usage. The mode-of-action screening approach advances scientific understanding and is preferred over other options based on apical tests, as these essentially are reproductive effects screens that are not necessarily specific for endocrine activity. Because Tier II tests include the critical apical endpoints used in the pubertal models, a mode-of-action approach provides complementary rather than redundant data. By identifying the potential mode of action, critical endpoints can be included in Tier II studies that will be used to define dose-response curves and no observed adverse effect levels (NOAELs)/no observed effect levels (NOELs) for the compound.

Differential alteration by thalidomide of the glutathione content of rat vs. rabbit conceptuses in vitro

Thalidomide has been shown to cause limb reduction defects in rabbits with much greater potency than in rats, possibly due to inherent biochemical differences between the two species. Whole embryo culture was used to make direct comparisons between thalidomide-sensitive New Zealand White rabbits and thalidomide-resistant Sprague-Dawley rats, focusing on the possible roles of glutathione (GSH) and cysteine in mechanisms of thalidomide teratogenicity. Conceptuses were treated by adding thalidomide (0, 5, 15, and 30 microM) directly to the culture media containing conceptuses of similar gestational stages. Embryos and visceral yolk sacs (VYS) were measured for changes in GSH and cysteine content using HPLC after 24 h of exposure in vitro. Thalidomide-induced (15 and 30 microM) depletion of VYS GSH occurred only in the rabbit, where GSH concentrations (pmol/microg protein) fell significantly to about 50% of control. Rat VYS did not show a significant GSH depletion at any thalidomide concentration tested. Comparison between species showed that the control rabbit VYS contained 35% less GSH than the control rat VYS. Control rat embryos and control rabbit embryos contained similar concentrations of GSH, but thalidomide treatment preferentially depleted GSH in the rabbit at lower thalidomide concentrations (5 micro/M). Cysteine concentrations were not significantly altered from control in the embryo or VYS of either species when treated with thalidomide. However, although control cysteine concentrations did not differ significantly between rat and rabbit VYS, control cysteine levels in rabbit embryos were 65% lower than those in control rat embryos. Rabbit conceptuses displayed lower species-specific GSH and cysteine levels and a greater propensity for thalidomide-induced GSH depletion than in rat conceptuses, consistent with the greater sensitivity of the rabbit to thalidomide teratogenicity. These thalidomide-induced and inherent species differences implicate a possible role for GSH and redox status in the mechanisms of thalidomide teratogenicity.

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

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.

Differential antioxidant enzyme activities and glutathione content between rat and rabbit conceptuses

Redox status regulates numerous cellular processes like transcription factor activation and binding, protein folding, and calcium sequestration. Because the most abundant reducing equivalent in the cell is glutathione (GSH), it could play a role for teratogens that cause oxidative stress and disrupt pathways involved in differentiation and proliferation. Investigation of the redox status of two species that have demonstrated differential sensitivity to teratogens represents a novel approach for determining the role of redox alteration in teratogenesis. Furthermore, examining specific regions of the embryo may also help to explain why certain tissues are uniquely sensitive, while others are resistant to oxidative insult. In the presented study, New Zealand White rabbit (GD 12) and Sprague Dawley rat embryos (GD 13) were removed from the uterus on days of similar development. Each embryo was dissected into three portions-the limbs, the head, and the trunk. Samples were placed in the appropriate buffers for the measurement of both direct and indirect redox status contributors-GSH, cysteine, thioredoxin, glutathione disulfide, protein-glutathione mixed disulfides, superoxide dismutase, glutathione peroxidase, and glutathione disulfide reductase. Species comparison of whole embryos indicated that the rabbit embryo possesses a higher redox potential (more oxidative) than the rat embryo. Findings, in general, show that the rabbit may be more sensitive to redox-altering teratogens because it is inherently more pro-oxidizing and may be more easily perturbed resulting in misregulation of cellular processes. Differences were most apparent in the limb as compared to the embryonic head and trunk, where the rabbit limb has a significantly more pro-oxidizing redox environment than the rat limb. Species comparisons like these may help in the understanding of how redox shifts affect cellular processes and would contribute to regulation of biochemical and molecular events that may be associated with mechanisms of teratogenesis. These may contribute to a more complete rationale for choosing a species for study and provide a better correlation with human developmental toxicants.

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 microM) or DEM (5-25 microM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.

Mode of action: oxalate crystal-induced renal tubule degeneration and glycolic acid-induced dysmorphogenesis--renal and developmental effects of ethylene glycol.

Ethylene glycol can cause both renal and developmental toxicity, with metabolism playing a key role in the mode of action (MOA) for each form of toxicity. Renal toxicity is ascribed to the terminal metabolite oxalic acid, which precipitates in the kidney in the form of calcium oxalate crystals and is believed to cause physical damage to the renal tubules. The human relevance of the renal toxicity of ethylene glycol is indicated by the similarity between animals and humans of metabolic pathways, the observation of renal oxalate crystals in toxicity studies in experimental animals and human poisonings, and cases of human kidney and bladder stones related to dietary oxalates and oxalate precursors. High-dose gavage exposures to ethylene glycol also cause axial skeletal defects in rodents (but not rabbits), with the intermediary metabolite, glycolic acid, identified as the causative agent. However, the mechanism by which glycolic acid perturbs development has not been investigated sufficiently to develop a plausible hypothesis of mode of action, nor have any cases of ethylene glycol-induced developmental effects been reported in humans. Given this, and the variations in sensitivity between animal species in response, the relevance to humans of ethylene glycol-induced developmental toxicity in animals is unknown at this time.

Identification of proximate toxicant for ethylene glycol developmental toxicity using rat whole embryo culture.

The effects of ethylene glycol (EG) and its metabolite, glycolic acid (GA), were compared by culturing day 10.5 rat conceptuses for 46 h in media containing 0.5, 2.5, 12.5, 25 or 50 mM EG or GA. EG up to 50 mM was essentially without effect, whereas > or = 12.5 mM GA inhibited embryo growth and development. Craniofacial dysmorphogenesis was observed in 70% of the 12.5 mM GA embryos (0% in controls). To determine if GA toxicity in vitro was an indirect effect of medium acidification, embryos were cultured in 12.5 mM GA (pH 6.7), 12.5 mM sodium glycolate (pH 7.4), or in control medium (pH 7.4 or 6.7). The percentage of dysmorphic embryos was 67% for the 12.5 mM GA (pH 6.7) group, 58% for the sodium glycolate (pH 7.4) group, 8% in the pH 6.7 controls, and 0% in the pH 7.4 controls. These results suggest that GA, not parent EG, is the active toxicant for EG-induced developmental toxicity and that acidification of culture medium pH plays only a minor role in GAs effects in vitro. The identification of GA as the active toxicant is important for the risk assessment of EG because GA exhibits dose-rate-dependent, nonlinear kinetics in vivo.