George P. Daston

Identification and Characterization of Toxicity of Contaminants in Pet Food Leading to an Outbreak of Renal Toxicity in Cats and Dogs

This paper describes research relating to the major recall of pet food that occurred in Spring 2007 in North America. Clinical observations of acute renal failure in cats and dogs were associated with consumption of wet pet food produced by a contract manufacturer producing for a large number of companies. The affected lots of food had been formulated with wheat gluten originating from China. Pet food and gluten were analyzed for contaminants using several configurations of high-performance liquid chromatography (HPLC) and mass spectrometry (MS), which revealed a number of simple triazine compounds, principally melamine and cyanuric acid, with lower concentrations of ammeline, ammelide, ureidomelamine, and N-methylmelamine. Melamine and cyanuric acid, have been tested and do not produce acute renal toxicity. Some of the triazines have poor solubility, as does the compound melamine cyanurate. Pathological evaluation of cats and dogs that had died from the acute renal failure indicated the presence of crystals in kidney tubules. We hypothesized that these crystals were composed of the poorly soluble triazines, a melamine-cyanuric acid complex, or a combination. Sprague dawley rats were given up to 100 mg/kg ammeline or ammelide alone, a mixture of melamine and cyanuric acid (400/400 mg/kg/day), or a mixture of all four compounds (400 mg/kg/day melamine, 40 mg/kg/day of the others). Neither ammeline nor ammelide alone produced any renal effects, but the mixtures produced significant renal damage and crystals in nephrons. HPLC-MS/MS confirmed the presence of melamine and cyanuric acid in the kidney. Infrared microspectroscopy on individual crystals from rat or cat (donated material from a veterinary clinic) kidneys confirmed that they were melamine-cyanuric acid cocrystals. Crystals from contaminated gluten produced comparable spectra. These results establish the causal link between the contaminated gluten and the adverse effects and provide a mechanistic explanation for how two apparently innocuous compounds could have adverse effects in combination, that is, by forming an insoluble precipitate in renal tubules leading to progressive tubular blockage and degeneration.

Meeting Report: Hazard Assessment for Nanoparticles—Report from an Interdisciplinary Workshop

In this report we present the findings from a nanotoxicology workshop held 6–7 April 2006 at the Woodrow Wilson International Center for Scholars in Washington, DC. Over 2 days, 26 scientists from government, academia, industry, and nonprofit organizations addressed two specific questions: what information is needed to understand the human health impact of engineered nanoparticles and how is this information best obtained? To assess hazards of nanoparticles in the near-term, most participants noted the need to use existing in vivo toxicologic tests because of their greater familiarity and interpretability. For all types of toxicology tests, the best measures of nanoparticle dose need to be determined. Most participants agreed that a standard set of nanoparticles should be validated by laboratories worldwide and made available for benchmarking tests of other newly created nanoparticles. The group concluded that a battery of tests should be developed to uncover particularly hazardous properties. Given the large number of diverse materials, most participants favored a tiered approach. Over the long term, research aimed at developing a mechanistic understanding of the numerous characteristics that influence nanoparticle toxicity was deemed essential. Predicting the potential toxicity of emerging nanoparticles will require hypothesis-driven research that elucidates how physicochemical parameters influence toxic effects on biological systems. Research needs should be determined in the context of the current availability of testing methods for nanoscale particles. Finally, the group identified general policy and strategic opportunities to accelerate the development and implementation of testing protocols and ensure that the information generated is translated effectively for all stakeholders.

Environmental estrogens and reproductive health: A discussion of the human and environmental data

Estrogenic activity of certain xenobiotics is an established mechanism of toxicity that can impair reproductive function in adults of either sex, lead to irreversible abnormalities when administered during development, or cause cancer. The concern has been raised that exposure to ambient levels of estrogenic xenobiotics may be having widespread adverse effects on reproductive health of humans and wildlife. The purpose of this review is to evaluate (a) the nature of the evidence supporting this concern, and (b) the adequacy of toxicity screening to detect, and risk assessment procedures to establish safe levels for, agents acting by this mechanism. Observations such as adverse developmental effects after maternal exposure to therapeutic levels of the potent estrogen diethylstilbestrol or male fertility problems after exposure to high levels of the weak estrogen chlordecone clearly demonstrate that estrogenicity is active as a toxic mechanism in humans. High level exposures to estrogenic compounds have also been shown to affect specific wildlife populations. However, there is little direct evidence to indicate that exposures to ambient levels of estrogenic xenobiotics are affecting reproductive health. Reports of historical trends showing decreasing reproductive capacity (e.g., decreased sperm production over the last 50 years) are either inconsistent with other data or have significant methodologic inadequacies that hinder interpretation. More reliable historical trend data show an increase in breast cancer rate, but the most comprehensive epidemiology study to data failed to show an association between exposure to persistent, estrogenic organochlorine compounds and breast cancer. Clearly, more work needs to be done to characterize historical trends in humans and background incidence of abnormalities in wildlife populations, and to test hypotheses about ambient exposure to environmental contaminants and toxic effects, before conclusions can be reached about the extent or possible causes of adverse effects. It is unlikely that current lab animal testing protocols are failing to detect agents with estrogenic activity, as a wide array of estrogen-responsive endpoints are measured in standard testing batteries. Routine testing for aquatic and wildlife toxicity is more limited in this respect, and work should be done to assess the validity of applying mammalian toxicology data for submammalian hazard identification. Current risk assessment methods appear to be valid for estrogenic agents, although the database for evaluating this is limited. In conclusion, estrogenicity is an important mechanism of reproductive and developmental toxicity; however, there is little evidence at this point that low level exposures constitute a human or ecologic health risk. Given the potential consequences of an undetected risk, more research is needed to investigate associations between exposures and effects, both in people and animals, and a number of research questions are identified herein. The lack of evidence demonstrating widespread xenobiotic-induced estrogenic risk suggests that far-reaching policy decisions can await these research findings.

The Plausibility of Micronutrient Deficiencies Being a Significant Contributing Factor to the Occurrence of Pregnancy Complications

Numerous studies support the concept that a major cause of pregnancy complications can be suboptimal embryonic and fetal nutrition. Although the negative effects of diets low in energy on pregnancy outcome are well documented, less clear are the effects of diets that are low in one or more essential micronutrients. However, several observational and intervention studies suggest that diets low in essential vitamins and minerals can pose a significant reproductive risk in diverse human populations. Although maternal nutritional deficiencies typically occur as a result of low dietary intakes of essential nutrients, nutritional deficiencies at the level of the conceptus can arise through multiple mechanisms. Evidence from experimental animals supports the concept that in addition to primary deficiencies, secondary embryonic and fetal nutritional deficiencies can be caused by diverse factors including genetics, maternal disease, toxicant insults and physiological stressors that can trigger a maternal acute phase response. These secondary responses may be significant contributors to the occurrence of birth defects. An implication of the above is that the frequency and severity of pregnancy complications may be reduced through an improvement in the micronutrient status of the mother.

Leydig cell hyperplasia and adenoma formation: Mechanisms and relevance to humans

Leydig cell adenomas are observed frequently in studies evaluating the chronic toxicity of chemical agents in laboratory animals. Doubts have been raised about the relevance of such responses for human risk assessment, but the question of relevance has not been evaluated and presented in a comprehensive manner by a broad group of experts. This article reports the consensus conclusions from a workshop on rodent Leydig cell adenomas and human relevance. Five aspects of Leydig cell biology and toxicology were discussed: 1) control of Leydig cell proliferation; 2) mechanisms of toxicant-induced Leydig cell hyperplasia and tumorigenesis; 3) pathology of Leydig cell adenomas; 4) epidemiology of Leydig cell adenomas; and 5) risk assessment for Leydig cell tumorigens. Important research needs also were identified. Uncertainty exists about the true incidence of Leydig cell adenomas in men, although apparent incidence is rare and restricted primarily to white males. Also, surveillance databases for specific therapeutic agents as well as nicotine and lactose that have induced Leydig cell hyperplasia or adenoma in test species have detected no increased incidence in humans. Because uncertainties exist about the true incidence in humans, induction of Leydig cell adenomas in test species may be of concern under some conditions. Occurrence of Leydig cell hyperplasia alone in test species after lifetime exposure to a chemical does not constitute a cause for concern in a risk assessment for carcinogenic potential, but early occurrence may indicate a need for additional testing. Occurrence of Leydig cell adenomas in test species is of potential concern as both a carcinogenic and reproductive effect if the mode of induction and potential exposures cannot be ruled out as relevant for humans. The workgroup focused on seven hormonal modes of induction of which two, GnRH agonism and dopamine agonism, were considered not relevant to humans. Androgen receptor antagonism, 5 alpha-reductase inhibition, testosterone biosynthesis inhibition, aromatase inhibition, and estrogen agonism were considered to be relevant or potentially relevant, but quantitative differences may exist across species, with rodents being more sensitive. A margin of exposure (MOE; the ratio of the lowest exposure associated with toxicity to the human exposure level) approach should be used for compounds causing Leydig cell adenoma by a hormonal mode that is relevant to humans. For agents that are positive for mutagenicity, the decision regarding a MOE or linear extrapolation approach should be made on a case-by-case basis. In the absence of information about mode of induction, it is necessary to utilize default assumptions, including linear behavior below the observable range. All of the evidence should be weighed in the decision-making process.

Effect of copper deficiency on prenatal development and pregnancy outcome.

Copper deficiency during embryonic and fetal development can result in numerous gross structural and biochemical abnormalities. Such a deficiency can arise through a variety of mechanisms, including low maternal dietary copper intake, disease-induced or drug-induced changes in maternal and conceptus copper metabolism, or both. These issues are discussed in this article along with the use of in vitro embryo culture models to study the mechanisms underlying copper deficiency-induced teratogenesis. Current data suggest that changes in free radical defense mechanisms, connective tissue metabolism, and energy production can all contribute to the dysmorphogenesis associated with developmental copper deficiency.

Perspectives on validation of high-throughput assays supporting 21st century toxicity testing

In vitro high-throughput screening (HTS) assays are seeing increasing use in toxicity testing. HTS assays can simultaneously test many chemicals but have seen limited use in the regulatory arena, in part because of the need to undergo rigorous, time-consuming formal validation. Here we discuss streamlining the validation process, specifically for prioritization applications. By prioritization, we mean a process in which less complex, less expensive, and faster assays are used to prioritize which chemicals are subjected first to more complex, expensive, and slower guideline assays. Data from the HTS prioritization assays is intended to provide a priori evidence that certain chemicals have the potential to lead to the types of adverse effects that the guideline tests are assessing. The need for such prioritization approaches is driven by the fact that there are tens of thousands of chemicals to which people are exposed, but the yearly throughput of most guideline assays is small in comparison. The streamlined validation process would continue to ensure the reliability and relevance of assays for this application. We discuss the following practical guidelines: (1) follow current validation practice to the extent possible and practical; (2) make increased use of reference compounds to better demonstrate assay reliability and relevance; (3) de-emphasize the need for cross-laboratory testing; and (4) implement a web-based, transparent, and expedited peer review process.

Application of Key Events Analysis to Chemical Carcinogens and Noncarcinogens

The existence of thresholds for toxicants is a matter of debate in chemical risk assessment and regulation. Current risk assessment methods are based on the assumption that, in the absence of sufficient data, carcinogenesis does not have a threshold, while noncarcinogenic endpoints are assumed to be thresholded. Advances in our fundamental understanding of the events that underlie toxicity are providing opportunities to address these assumptions about thresholds. A key events dose-response analytic framework was used to evaluate three aspects of toxicity. The first section illustrates how a fundamental understanding of the mode of action for the hepatic toxicity and the hepatocarcinogenicity of chloroform in rodents can replace the assumption of low-dose linearity. The second section describes how advances in our understanding of the molecular aspects of carcinogenesis allow us to consider the critical steps in genotoxic carcinogenesis in a key events framework. The third section deals with the case of endocrine disrupters, where the most significant question regarding thresholds is the possible additivity to an endogenous background of hormonal activity. Each of the examples suggests that current assumptions about thresholds can be refined. Understanding inter-individual variability in the events involved in toxicological effects may enable a true population threshold(s) to be identified.

Altered maternal zinc metabolism following exposure to diverse developmental toxicants

It has been hypothesized that one mechanism contributing to the developmental toxicity of some xenobiotics is an embryonic/fetal zinc (Zn) deficiency that occurs secondary to toxicant-induced changes in maternal Zn metabolism. We studied the influence of diverse toxicants (urethane, ethanol, melphalan, arsenic, and alpha-hederin) on maternal-embryonic Zn metabolism and maternal liver metallothionein (MT) induction in Sprague-Dawley rats given a 65Zn-labelled meal by gavage 8 h after toxicant exposure and killed 10 h later on gestation day 12.5. Exposure to the toxicants resulted in increases in maternal hepatic MT concentrations that generally exceeded that which could be accounted for by reductions in food intake. 65Zinc retention was higher in maternal liver and lower in the products of conception in the toxicant-exposed groups. Strong linear relationships were found; as maternal liver MT concentrations increased, 65Zn retention in maternal liver was increased and 65Zn distribution to the conceptuses was decreased. These results support the hypothesis that diverse insults can produce developmental toxicity, in part, by altering maternal and embryonic Zn metabolism.

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.