In Vitro Developmental Toxicology Screens: A Report on the Progress of the Methodology and Future Applications.

There has been increasing focus on generation and assessment of in vitro developmental toxicology models for assessing teratogenic liability of chemicals. The driver for this focus has been to find reliable in vitro assays that will reduce or replace the use of in vivo tests for assessing teratogenicity. Such efforts may be eventually applied in testing pharmaceutical agents where a developmental toxicology assay or battery of assays may be incorporated into regulatory testing to replace one of the two species currently used in teratogenic assessment. Such assays may be eventually applied in testing a broader spectrum of chemicals, supporting efforts aligned with Tox21 strategies and responding to REACH legislation. This review describes the developmental toxicology assays that are of focus in these assessments: rodent whole embryo culture, zebrafish embryo assays, and embryonic stem cell assays. Progress on assay development as well as future directions of how these assays are envisioned to be applied for broader safety testing of chemicals are discussed. Altogether, the developmental model systems described in this review provide rich biological systems that can be utilized in better understanding teratogenic mechanisms of action of chemotypes and are promising in providing proactive safety assessment related to developmental toxicity. Continual advancements in refining/optimizing these in vitro assays are anticipated to provide a robust data set to provide thoughtful assessment of how whole animal teratogenicity evaluations can be reduced/refined in the future.

Development and characterization of rat duodenal organoids for ADME and toxicology applications.

Many in vitro gastrointestinal models have been developed with the hope that they will continue to improve in their similarity to the organs from which they were isolated. Intestinal organoids isolated from various species are now being used to investigate physiology and pathophysiology. In this study, intestinal stem cells were isolated from adult rat duodenum and culture conditions were optimized to promote the growth, differentiation and development of 3D organoids. We optimized and characterized rat duodenal organoids with light and electron microscopy, immunofluorescence and notably, global mRNA expression. The metabolic capacity of these cultures was investigated using probe substrates for multiple phase I and phase II drug metabolizing enzymes and found to be in line with previous results from intestinal primary cultures and a significant improvement over immortalized cell lines. Over the course of differentiation, the gene expression profiles of the rat duodenal organoids were consistent with expected trends in differentiation to various cell lineages reflecting the duodenum in vivo. Further, incubations of these cultures with naproxen and celecoxib resulted in cytotoxicity consistent with the direct cytotoxic effects of these drugs to duodenum in vivo. Based on these characteristics, the rat duodenal organoids described herein will provide a novel platform for investigating a wide variety of mechanistic questions.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

The rat whole embryo culture assay using the Dysmorphology Score system.

The rat whole embryo culture (WEC) system has been used extensively for characterizing teratogenic properties of test chemicals. In this chapter, we describe the methodology for culturing rat embryos as well as a new morphological score system, the Dysmorphology Score (DMS) system for assessing morphology of mid gestation (gestational day 11) rat embryos. In contrast to the developmental stage focused scoring associated with the Brown and Fabro score system, this new score system assesses the respective degree of severity of dysmorphology, which delineates normal from abnormal morphology of specific embryonic structures and organ systems. This score system generates an approach that allows rapid identification and quantification of adverse developmental findings, making it conducive for characterization of compounds for teratogenic properties and screening activities.