Selecting appropriate animal models and experimental designs for endocrine disruptor research and testing studies

Evidence that chemicals in the environment may cause developmental and reproductive abnormalities in fish and wildlife by disrupting normal endocrine functions has increased concern about potential adverse human health effects from such chemicals. US laws have now been enacted that require the US Environmental Protection Agency (EPA) to develop and validate a screening program to identify chemicals in food and water with potential endocrine-disrupting activity. EPA subsequently proposed an Endocrine Disruptor Screening Program that uses in vitro and in vivo test systems to identify chemicals that may adversely affect humans and ecologically important animal species. However, the endocrine system can be readily modulated by many experimental factors, including diet and the genetic background of the selected animal strain or stock. It is therefore desirable to minimize or avoid factors that cause or contribute to experimental variation in endocrine disruptor research and testing studies. Standard laboratory animal diets contain high and variable levels of phytoestrogens, which can modulate physiologic and behavioral responses similar to both endogenous estrogen as well as exogenous estrogenic chemicals. Other studies have determined that some commonly used outbred mice and rats are less responsive to estrogenic substances than certain inbred mouse and rat strains for various estrogen-sensitive endpoints. It is therefore critical to select appropriate biological models and diets for endocrine disruptor studies that provide optimal sensitivity and specificity to accomplish the research or testing objectives. An introduction is provided to 11 other papers in this issue that review these and other important laboratory animal experimental design considerations in greater detail, and that review laboratory animal and in vitro models currently being used or evaluated for endocrine disruptor research and testing. Selection of appropriate animal models and experimental design parameters for endocrine disruptor research and testing will minimize confounding experimental variables, increase the likelihood of replicable experimental results, and contribute to more reliable and relevant test systems.     

Integrated decision-tree testing strategies for developmental and reproductive toxicity with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME conducted a research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for the use of alternative methods (both in vitro and in silico) in developmental and reproductive toxicity testing. It considers many tests based on primary cells and cell lines, and the available expert systems and QSARs for developmental and reproductive toxicity, and also covers tests for endocrine disruption. Ways in which reduction and refinement measures can be used are also discussed, particularly the use of an enhanced one-generation reproductive study, which could potentially replace the two-generation study, and therefore considerably reduce the number of animals required in reproductive toxicity. Decision-tree style integrated testing strategies are also proposed for developmental and reproductive toxicity and for endocrine disruption, followed by a number of recommendations for the future facilitation of developmental and reproductive toxicity testing, with respect to human risk assessment.     

Establishment and Assessment of a New Human Embryonic Stem Cell‐Based Biomarker Assay for Developmental Toxicity Screening

A metabolic biomarker‐based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery‐phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure‐based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9‐point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.     

Sea urchin development: an alternative model for mechanistic understanding of neurodevelopment and neurotoxicity

Echinoderm early developmental stages might supply a good tool for toxicity testing in different fields, ranging from environment to food contamination, and in full respect of the 3Rs objectives (reduction, refinement, and replacement of animal experiments) that will eventually lead to the replacement of high vertebrate animal testing in toxicology. Sea urchin is one of the few organismic models considered by the European Agency for Alternative models. Actually, sea urchin embryonic development has been studied for over a century, and the complex nets of intercellular communications leading to the different events are well known, as well the possibility for environmental molecules and their residuals to interfere with such communications, causing developmental anomalies. In particular, the main goal of toxicologists since several years has been to establish a correlation between the cell-to-cell communications occurring during different developmental events and the signals occurring during neurogenesis, with the aim to pursue a mechanistic understanding of these processes and their deviations caused by stressors from different sources.     

Mechanistic considerations in small fish carcinogenicity testing

Historically, small fish species have proven useful both as environmental sentinels and as versatile test animals in toxicity and carcinogenicity bioassays. They can be bred in large numbers, have low maintenance and bioassay costs, and have a low background incidence of tumors. However, more mechanistic information is needed to help validate the information garnered from these models and to keep pace with other more fully developed animal models. This paper focuses on mechanistic considerations when using small fish models for carcinogenicity testing. Several small aquarium fish species have proven useful. The Japanese medaka is perhaps the best characterized small fish model for carcinogenicity testing; however, the zebrafish is emerging as an important model because it is well characterized genetically. Both route and methodology of exposure may affect the outcome of the study. Most studies have been conducted by introducing the test compound into the ambient water, but dietary exposures and embryo microinjection have also been used. Other considerations in study design include use of an initiating carcinogen, such as diethlynitrosamine, and differences in xenobiotic metabolism, such as the fact that fish CYP2B is refractory to phenobarbital induction. The small size of these models has perhaps limited some types of mechanistic studies, such as formation and repair of DNA adducts in response to carcinogen exposure. However, improved analytical methods are allowing greater resolution and should be applied to small fish species. Slide-based methods such as immunohistochemistry are an important adjunct to routine histopathology and should be included in study design. However, there is a need for development of more species-specific antibodies for fish research. There is also a need for more fish-specific data on cytokines, serum biochemistry, and oncogenes to strengthen the use of these important test models.     

Understanding dioxin developmental toxicity using the zebrafish model

Zebrafish (Danio rerio) have advantages over mammals as an animal model for investigating developmental toxicity. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin, TCDD), a persistent global contaminant, is the most comprehensively studied developmental toxicant in zebrafish. The hallmark responses of TCDD developmental toxicity manifested in zebrafish larvae include edema, anemia, hemorrhage, and ischemia associated with arrested growth and development. Heart and vasculature development and function are severely impaired, and jaw malformations occur secondary to inhibited chondrogenesis. The swim bladder fails to inflate, and the switch from embryonic to adult erythropoiesis is blocked. This profile of developmental toxicity responses, commonly referred to as "blue sac syndrome" because the edematous yolk sac appears blue, is observed in the larval form of all freshwater fish species exposed to TCDD at the embryonic stage of development. Components of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AHR/ARNT) signaling pathway in zebrafish have been identified and functionally characterized. Their role in mediating TCDD toxicity has been determined using morpholinos to specifically knockdown the translation of zfAHR1, zfAHR2, zfARNT1, and zfARNT2 mRNAs, respectively, and a line of zfARNT2 null mutant zebrafish has provided further insight. These studies have shown that zfAHR2 and zfARNT1 mediate TCDD developmental toxicity. In addition, the growing use of molecular and genomic tools for research on zebrafish have led to advances in our understanding of the mechanism of TCDD developmental toxicity at the molecular level, including the recent finding that toxicity is not mediated by increased cytochrome P4501A (zfCYP1A) expression.     

Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro

There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.     

Generating new marine cell lines and transgenic species--conference summary

Marine species offer a tremendous diversity of life histories, physiologies, genetics, behaviors, and biologies, reflecting myriad adaptations to the water environment. Historically, marine vertebrates, particularly fish, have played significant roles in a wide range of disciplines, including environmental toxicology, genetics, developmental biology, and physiology, among others. Much still remains to be learned from these animals, and there is a growing need for new marine models. Models for expression of marine animal genes have been limited to heterologous expression systems. While there is still a great deal to gain from heterologous expression systems, the interactions of genes with one another can best be determined in homologous expression systems where appropriate interactions are possible. This has become particularly important with the development of functional genomics in marine models. These homologous gene expression systems will be key to the use of functional genomics for marine animal molecular physiology and toxicology.     

Developmental toxicology: new directions workshop: refining testing strategies and study designs

In April 2009, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute's (HESI) Developmental and Reproductive Toxicology Technical Committee held a two-day workshop entitled "Developmental Toxicology-New Directions." The third session of the workshop focused on ways to refine animal studies to improve relevance and predictivity for human risk. The session included five presentations on: (1) considerations for refining developmental toxicology testing and data interpretation; (2) comparative embryology and considerations in study design and interpretation; (3) pharmacokinetic considerations in study design; (4) utility of genetically modified models for understanding mode-of-action; and (5) special considerations in reproductive testing for biologics. The presentations were followed by discussion by the presenters and attendees. Much of the discussion focused on aspects of refining current animal testing strategies, including use of toxicokinetic data, dose selection, tiered/triggered testing strategies, species selection, and use of alternative animal models. Another major area of discussion was use of non-animal-based testing paradigms, including how to define a "signal" or adverse effect, translating in vitro exposures to whole animal and human exposures, validation strategies, the need to bridge the existing gap between classical toxicology testing and risk assessment, and development of new technologies. Although there was general agreement among participants that the current testing strategy is effective, there was also consensus that traditional methods are resource-intensive and improved effectiveness of developmental toxicity testing to assess risks to human health is possible. This article provides a summary of the session's presentations and discussion and describes some key areas that warrant further consideration.     

An interspecies comparison of placental antibody transfer: New insights into developmental toxicity testing of monoclonal antibodies

There are profound differences in maternofetal transfer of immunoglobulins between species with extensive gestational transfer of maternal immunoglobulins in primates (including humans) via the chorioallantoic placenta as well as in rabbits and guinea pigs via the inverted yolk sac splanchnopleure. In contrast, other neonatal rodents (rats and mice) receive passive immunity predominantly postnatally. This transfer is mediated principally via FcRn receptors. Therapeutic monoclonal antibodies (mAbs) are most commonly of the IgG1 subclass, which is transported most efficiently to the fetus. In all animal species used for testing developmental toxicity, fetal exposure to IgG is very low during organogenesis, but this increases during the latter half of gestation such that the neonate is born with an IgG1 concentration similar to the mother (but not rats and mice). Review of mAb developmental toxicity studies of licensed products reveals Cynomolgus monkey as the species used in the majority of the cases (10 out of 15). Pregnancy outcome data from women gestationally exposed to mAb is limited. In general, the findings are consistent with the expected low exposure during organogenesis. Guinea-pigs and rabbits are potential candidates as “alternatives” to the use of nonhuman primates as the maternofetal transfer in the last part of gestation is at a level similar in humans. Based on the pattern of placental transfer of IgG in humans, study designs that allow detection of both the indirect effects in early gestation plus the effects of direct fetal exposure in mid and late gestation are recommended for developmental toxicity of mAbs. Birth Defects Res (Part B), 86:328–344, 2009. © 2009 Wiley-Liss, Inc.     

Setting Pesticide Reference Doses: A Retrospective Analysis Examining Key Data and Choices

Toxicity tests are widely used to set “acceptable” levels of chemical exposure. Different organizations have identified a base set of tests specifying a mix of endpoints, durations, and species to be tested. A specific test and endpoint is chosen as the basis for calculation of human health risk values like reference doses (RfDs). This study empirically evaluates the data and choices made in setting acute and chronic RfDs for 352 conventional pesticides. The results suggest that for Acute, Acute-Female Specific, and Chronic RfDs one test is used far more than others. Ninety-six percent of the 116 Acute Female-Specific RfDs relied on a developmental toxicity test and 78% of Chronic RfDs used the chronic bioassay. Tests in rats were used far more often than other species in all RfD calculations. For all types of RfDs a total uncertainty factor of 100 was most common although values as low as 1 and as high as 3000 were seen. These results provide insights not only into the science policy frameworks used, but also into ways toxicity testing and risk assessment may be streamlined and made more efficient.     

The luminescent bacteria toxicity test: Its potential as an in vitro alternative

During the past several years, the use of animals for toxicity testing has come under critical surveillance. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives for some of the whole animal toxicity assays. One assay proposed as an alternative to animal testing is the luminescent bacteria toxicity test (LBT), provided under the trade name of Microtox®. The sensitivity and specificity of the LBT was compared with two commonly used toxicity tests–-the L-929 Minimal Eùgle's Medium (MEM) elution cytotoxicity test and the Draize test. Cytotoxicity and LBT test data from 709 medical device and biomaterial extracts were compared using a positive/negative ranking system which provided a measurement of false positive and false negative results. These data were compiled from nine separate laboratories producing or using a wide variety of biomaterials and medical device products. The LBT was more sensitive than the tissue culture assay and displayed few false negatives. LBT EC₅₀ values were compared with eye irritancy categories for a group of 34 chemicals and 27 personal care products. As with tissue culture, the LBT was more sensitive and produced minimal false negatives. The data from this study indicate the LBT has potential as a rapid, simple method to screen biomaterials and personal care products for toxicity and irritancy.     

Activity profiles of developmental toxicity: design considerations and pilot implementation

The available literature was searched for quantitative test results from both in vitro and in vivo assays for developmental toxicity for five model compounds: cyclophosphamide, methotrexate, hydroxyurea, caffeine, and ethylenethiourea. These compounds were chosen on the basis of their extensive utilization in a variety of assay systems for developmental toxicity as evidenced by their representation in the ETIC database (each generally has 100-500 citations encompassing multiple test systems). Nine cellular-based assays, six assays using whole embryos in culture, as well as Segment II and abbreviated exposure tests for mammalian test species are included in the database. For each assay, the critical endpoints were identified, each of which was then provided a three-letter code, and the criteria for extraction of quantitative information were established. The extracted information was placed into a computerized reference file and subsequently plotted such that the qualitative (positive/negative) and quantitative (e.g., IC50, highest ineffective dose (HID), lowest effective dose (LED] results across all test systems could be displayed. The information contained in these profiles can be used to compare qualitative and quantitative results across multiple assay systems, to identify data gaps in the literature, to evaluate the concordance of the assays, to calculate relative potencies, and to examine structure-activity relationships.     

Proteomic analysis and in vitro developmental toxicity tests for mechanism-based safety evaluation of chemicals

Mechanism-based safety evaluation and reduction of animal use are important issues in recent developmental toxicology. In vitro developmental toxicity tests with proteomic analysis are the most promising solution to these issues. Groebe et al. systematically applied proteomic analysis to the embryonic stem cell test, a validated in vitro developmental toxicity test, and found protein-expression changes induced by model test chemicals selected from various categories of toxicity. Cluster analysis of all the proteins with expression changes classified the test chemicals into two groups: highly embryotoxic chemicals and non- or weakly embryotoxic chemicals. In addition, some protein biomarker candidates that were known to be involved in normal development were identified. Although further mechanistic investigations are needed, the use of in vitro developmental toxicity tests with proteomic analysis will contribute to mechanism-based safety evaluation with minimal use of animals.     

The guinea pig as an animal model for developmental and reproductive toxicology studies

BACKGROUND : Regulatory guidelines for developmental and reproductive toxicology (DART) studies require selection of “relevant” animal models as determined by kinetic, pharmacological, and toxicological data. Traditionally, rats, mice, and rabbits are the preferred animal models for these studies. However, for test articles that are pharmacologically inactive in the traditional animal models, the guinea pig may be a viable option. This choice should not be made lightly, as guinea pigs have many disadvantages compared to the traditional species, including limited historical control data, variability in pregnancy rates, small and variable litter size, long gestation, relative maturity at birth, and difficulty in dosing and breeding. METHODS : This report describes methods for using guinea pigs in DART studies and provides results of positive and negative controls. Standard study designs and animal husbandry methods were modified to allow mating on the postpartum estrus in fertility studies and were used for producing cohorts of pregnant females for developmental studies. RESULTS : A positive control study with the pregnancy-disrupting agent mifepristone resulted in the anticipated failure of embryo implantation and supported the use of the guinea pig model. Control data for reproductive endpoints collected from 5 studies are presented. CONCLUSION : In cases where the traditional animal models are not relevant, the guinea pig can be used successfully for DART studies. Birth Defects Res (Part B)86: 92-97, 2009. © 2009 Wiley-Liss, Inc.     

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture

BACKGROUND: The rodent whole embryo culture (WEC) system is a well‐established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis‐stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage‐specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure‐specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ‐selective effects. METHOD: This study describes a distinct morphological score system called the “Dysmorphology Score System (DMS system)” that has been developed for assessing gestation day 11 (approximately 20–26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. RESULT AND CONCLUSION: The DMS system enhances capabilities to rank‐order compounds based upon teratogenic potency, conduct structure‐ relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens. Birth Defects Res (Part B) 89:485–492, 2010. © 2010 Wiley‐Liss, Inc.     

Current and future needs for developmental toxicity testing

A review is presented of the use of developmental toxicity testing in the United States and international regulatory assessment of human health risks associated with exposures to pharmaceuticals (human and veterinary), chemicals (agricultural, industrial, and environmental), food additives, cosmetics, and consumer products. Developmental toxicology data are used for prioritization and screening of pharmaceuticals and chemicals, for evaluating and labeling of pharmaceuticals, and for characterizing hazards and risk of exposures to industrial and environmental chemicals. The in vivo study designs utilized in hazard characterization and dose-response assessment for developmental outcomes have not changed substantially over the past 30 years and have served the process well. Now there are opportunities to incorporate new technologies and approaches to testing into the existing assessment paradigm, or to apply innovative approaches to various aspects of risk assessment. Developmental toxicology testing can be enhanced by the refinement or replacement of traditional in vivo protocols, including through the use of in vitro assays, studies conducted in alternative nonmammalian species, the application of new technologies, and the use of in silico models. Potential benefits to the current regulatory process include the ability to screen large numbers of chemicals quickly, with the commitment of fewer resources than traditional toxicology studies, and to refine the risk assessment process through an enhanced understanding of the mechanisms of developmental toxicity and their relevance to potential human risk. As the testing paradigm evolves, the ability to use developmental toxicology data to meet diverse critical regulatory needs must be retained.     

Lysophospholipids in development: Miles apart and edging in

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids that participate in the regulation of mammalian cell proliferation, apoptosis, migration, and angiogenesis. These processes are each critical for successful embryogenesis, raising the possibility that lysophospholipid signaling may contribute to normal animal development. In fact, recent studies in developmental model systems have established that S1P and LPA are necessary for diverse developmental programs including those required for morphogenesis of vertebrate reproductive, cardiovascular and central and peripheral nervous systems (PNS), as well as the establishment of maternal-fetal circulation and the immune system. Genetic, morphological, and biochemical characterization of developmental model systems offer powerful approaches to elucidating the molecular mechanisms of lysophospholipid signaling and its contributions to animal development and postnatal physiology. In this review, the routes of S1P and LPA metabolism and our current understanding of lysophospholipid-mediated signal transduction in mammalian cells will be summarized. The evidence implicating lysophospholipid signaling in the development of specific vertebrate systems will then be reviewed, with an emphasis on signals mediated through G protein-coupled receptors of the Edg family. Lastly, recent insights derived from the study of simple metazoan models and implications regarding lysophospholipid signaling in organisms in which Edg receptors are not conserved will be explored.     

Organoid models of the tumor microenvironment and their applications

A small percentage of data obtained from animal/2D culture models can be translated to humans. Therefore, there is a need to using native tumour microenvironment mimicking models to improve preclinical screening and reduce this attrition rate. For this purpose, currently, the utilization of organoids is expanding. Tumour organoids can recapitulate tumour microenvironment that is including cancer cells and non-neoplastic host components. Indeed, tumour organoids, both phenotypically and genetically, resemble the tumour tissue that originated from it. The unique properties of the tumour microenvironment can significantly affect drug response and cancer progression. In this review, we will discuss about various organoid culture strategies for modelling the tumour immune microenvironment, their applications and advantages in cancer research such as testing cancer immunotherapeutics, developing novel approaches for personalized medicine, testing drug toxicity, drug screening, study cancer initiation and progression, and we will also review the limitations of organoid culture systems.