In Vitro Developmental Toxicology Screens: A Report on the Progress

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In Vitro Developmental Toxicology Screens: A Report on Progress of the Methodology and Future Applications Cindy X. Zhang, Jonathan Ball, Julie Panzica-Kelly, and Karen Augustine-Rauch Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00458 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 20, 2016

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Chemical Research in Toxicology

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

Cindy Zhang*, Jonathan Ball^, Julie Panzica-Kelly*, Karen Augustine-Rauch* *Discovery Toxicology, Bristol Myers Squibb ^University of Exeter, College of Life and Environmental Sciences, Exeter, UK Corresponding author information: Karen Augustine-Rauch, Ph.D. Research Fellow Discovery Toxicology Bristol-Myers Squibb Hopewell Site Bld 17, Room 233 311 Pennington-Rocky Hill Rd. Pennington, N.J. 08534

Telephone: (609)818-3406 [email protected]

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Table of Contents Graphic:

Characterize and Select Developmental Models Applications: Pharmaceutical and Chemical Testing, Animal Replacement Strategies

Generate In Vitro Teratology Screens

Validate and Maximize Assay Performance


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Abstract 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 2 species currently used in teratogenic assessment. Such assays may be eventually applied in testing 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 actions of chemotypes and are promising in providing proactive safety assessment related to developmental toxicity.

Continual advancements in

refining/optimizing these in vitro assays is anticipated to provide a robust data set to provide thoughtful assessment of how whole animal teratogenicity evaluations can be reduced/refined in the future.



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1. Introduction The high cost of regulatory reproductive toxicology testing typically positions the studies to be run relatively late in development, usually starting around a year prior to enrolment of women of child bearing potential into clinical trials (Phase III or late Phase II). The impact of pipeline attrition related to unexpected teratogenicity is potentially significant especially with pipelines that have therapeutic areas that include large populations of women of child bearing potential in the clinical populations such as migraine, obesity and anti-depressant indications. Substantial resources in compound, labor, time and animal use is associated with the studies to progress the compounds through the pipeline prior to the reproductive assessment. Furthermore, reproductive toxicology testing consists of a considerable number of studies to assess a compound’s

safety

profile

in

reproductive

(fertility/pre-implantation),

developmental

(teratogenicity) and post natal development, with teratogenicity assessment being typically conducted in two distinct species.

Overall, about 70% of the animals used in regulatory

industrial toxicology testing are related to supporting reproductive toxicology studies.1, 2 For these reasons, there has been broad interest in alternative methods to identify compounds with the potential to cause developmental toxicity ahead of the standard testing regime. Furthermore, there is a desire to consider alternative models to support regulatory testing in order to reduce animal usage.3, 4 This has led to substantial efforts in considering revisions to ICHS5 guidelines for regulatory reproductive toxicology where developmental toxicology assays (either individual or as a battery) are under consideration to be integrated into a testing paradigm that potentially supplements or defers in vivo regulatory testing.5-9

Although this evaluation continues,

considerable focus has been on reviewing applications of three developmental model systems for developmental toxicology assays: rodent whole embryo culture, zebrafish embryo and 4 ACS Paragon Plus Environment

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embryonic stem cells. We have previously published an overview of each of the respective assays and the current state of the science of assay development for teratogenicity screening in 2010.10 Since then, substantial work has been accomplished in making advances in these assays including approaches to further reduce animal use and performance testing. This report provides an update on the progress of these assays for teratogenicity screening and future applications as they apply to safety assessment of chemicals.

2. Rodent Whole Embryo Culture

Rodent whole embryo culture (WEC) has been used extensively for evaluating mechanisms of development and assessing teratogenic potential of compounds.11, 12 This in vitro system allows culture of early organogenesis-stage embryos intact in their visceral yolk sac for up to 72 hours, a critical embryo development stage that is sensitive to teratogenic insults (Figure 1). This stage in the rodent embryo covers critical morphogenic processes, which occur during the early part of the 1st trimester in human, such as neurulation and neural tube closure, neural crest migration, cardiac looping, facial morphogenesis, axial extension and early limb bud formation. This developmental stage is the most sensitive and critical window for teratogens. The system is easily manipulated allowing evaluation or treatment of embryos over the time course for assessing endpoints related to developmental or teratogenic mechanisms enabling evaluation of transcriptional, protein and epigenetic changes. Within the visceral yolk sac, the embryos grow within the amniotic membrane. Although small molecules can be easily added to the culture media, larger molecules (antibodies, oligonucleotides and other macromolecules) that may not sufficiently cross the visceral yolk sac can be delivered to the embryo by microinjection into the amniotic fluid. Finally, at the end of culture, embryos can be evaluated for overall



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embryo growth and a variety of morphological endpoints related to various structures and organ systems that undergo morphogenesis during this culture period. Altogether, the attributes of this developmental model system have made rodent embryo culture very conducive to studies evaluating mechanisms of normal and abnormal development as well as a system for evaluating teratogenic potential of compounds. A primary attribute of the use of WEC for screening teratogenic liability of compounds is that the rat represents the primary test species for in vivo teratology testing. In addition, there is a high level of cross-species conservation between rodent and human early organogenesis, enabling potential for risk assessment evaluations. Although the assay requires the use of rats, the approach aligns with reduction in animal use, as considerably fewer rats are required for running teratogenicity evaluation of a compound in a dose range in WEC (approximately 10 rats) vs. animal requirements for regulatory in vivo teratology testing (~88 rats for 3 doses and a vehicle control group).

In addition the WEC assay requires small amounts (1-10 mg) of

compound for testing. A drawback of the WEC system is that it does not reflect effects that may occur from maternal metabolism. However, the WEC system allows for well controlled parent compound exposure. Certain metabolites can be added to the WEC system at in vivo plasma concentration to match human-specific metabolites, so that drug induced teratogenic liabilities against pharmacokinetic differences between human and rodent test species can be better compared and assessed. However, the lack of maternal interactions can be a drawback, as maternal metabolites could directly affect embryo growth and ultimately lead to teratogenicity of embryos. Also, maternal environmental influences on embryonic development cannot be recapitulated in the WEC system. Additional limitations of the WEC system include that substantial training is 6 ACS Paragon Plus Environment

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required for learning the dissection methodology. In addition culture of rodent embryos can only be supported for a relatively short period, which may be sub-optimal for assessing drug effects on embryo development that occur in an earlier or later developmental stage. For instance, compounds that affect implantation or early gastrulation may not present adverse effects in WEC. In addition, alteration of fusion or closure of palatal shelves could result in cleft palate occurring at the more advanced stages of organogenesis, or skeletal development that happens in more advanced windows of organogenesis and fetal development.13, 14 Nevertheless, the WEC system has been widely utilized in various applications for mechanistic studies and characterization of teratogenic substances (reviewed in detail by Ellis-Hutchings and Carney 2010, AugustineRauch, et al 2010).10, 15 2.1 Progress in Developing WEC Screening Assays The earliest form of a WEC teratogenicity screen was established by the European Center for the Validation of Alternative Methods (ECVAM). This assay required morphological score assessment using the Brown and Fabro score system as a means to provide a standard for grading severity of adverse developmental effects in the mid-gestation rodent embryo.16 In this system, 17 morphological features were scored for presence of anatomical landmarks associated with developmental stage. The total score numbers from each individual embryo represented the overall morphological score.16 This WEC score system was integrated into a screening assay which was validated for inter-laboratory consistency by ECVAM and the European Teratology Society in 1996 and 2000. The ECVAM assay requires the IC50 value for cytotoxicity to be acquired from compound administration on NIH3T3 cells (postnatally derived fibroblasts). This metric serves as a surrogate value for “adult toxicity” that determines test concentrations for the WEC assay. The embryos treated with a series of test concentrations are evaluated for their 7 ACS Paragon Plus Environment


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growth and structural morphology using the comprehensive Brown and Fabro score system. During assay validation, six compounds were used to build a prediction model for classifying in vivo teratogenic liability, and fourteen other compounds that were non-teratogenic, weak teratogens and strong teratogens were evaluated by four laboratories to validate the prediction model using WEC assay. The overall predictivity of ECVAM validated prediction model was 80%.17, 18 To assess the reproducibility of ECVAM validated prediction model with extended number of chemicals, the WEC prediction model was challenged with 61 definitive animal teratogens and non teratogens. Among them, 48 were pharmaceutical compounds and 13 were original ECVAM tested chemicals. By applying the same linear discriminant prediction model used in EVACM validation study, the predictivity of 48 pharmaceutical compounds dropped to 56%, even though the predictivity of ECVAM’s original 13 chemicals was reproducible and reached to 77%. In an attempt to improve the predictivity, a different prediction model, Random Forest algorithm, was applied to 57 test chemicals to help identify the most useful endpoints and to provide a probability of correct classification. Unfortunately, improved predictivity could not be achieved.19 In addition to the challenge of working with a very complex prediction model, the score system used in the ECVAM WEC assay was comprehensive and low throughput. Although it provides a good quantitative approach to determine the stage of embryo developmental growth, the score system does not delineate structure-specific malformations induced by teratogenic insults. A rat embryo Dysmorphology Score (DMS) System was more recently generated that enhanced sensitivity to define the severity of structural malformations.20 The DMS system uses numerical scores to assess specific structures and organ systems and grade the severity of 8 ACS Paragon Plus Environment

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observed abnormalities (Table 1, Figure 2). This method enables identification of abnormalities, which may be subtle or limited to specific anatomical structures, enhancing overall sensitivity in identifying teratogenic insults as well as concentration relationship. Thus, the system can be applied to compound screening to delineate teratogenic potency and structure activity relationship of chemicals, which can be helpful in prioritizing compounds with less teratogenic characteristics, as well as identifying chemical moieties that may enhance teratogenicity. Working with group average morphological score data collected with the DMS score system, a statistical prediction model was developed to support a WEC experimental paradigm for compound screening that substantially conserved on animal use and animal-derived reagents (serum). In this study, approximately 30 compounds (definitive teratogens and non teratogens) were evaluated along a concentration range and the group average morphological score data was evaluated by a variety of statistical methods to determine whether there was an optimum concentration and set of morphological endpoints that best segregated teratogens from non teratogens.

Ultimately a simplified screen supported by a Recursive Partition model was

developed that required only one test concentration (1 uM) and assessment of 2 morphological group average scores (primitive spinal cord and heart) and 1 growth endpoint (group average somite number value).The prediction model was established using 59 test compounds, and achieved 83% concordance. The model was additionally evaluated by using 11 compounds that were not used to build the algorithm and 82% predictivity was achieved. In addition, 5-fold cross-validation was applied with all 70 compounds and achieved 74% predictivity.

This

prediction model has very similar predictivity compared to the more complex ECVAM prediction model, except much larger number of compounds were evaluated with higher throughput.20 Additionally the streamlined WEC screen eliminates the need to acquire IC50



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cytotoxicity values of a respective compound from a surrogate cell line culture to support concentration selection. Although promising as a teratogenicity screen, the streamlined WEC assay serves primarily as a hazard identification assay since only 1 test concentration is used to support the prediction model. Current activities associated with enhancing application of this assay involve testing compounds along concentration ranges that reflect in vivo Cmax exposures in animal studies that were non teratogenic as well as teratogenic. Such information is envisioned to be used in generating additional WEC-based assays that may have risk assessment applications, providing context regarding potential exposure margins that may be achieved in vivo.

3. Zebrafish Embryo Culture The tropical freshwater zebrafish (Danio rerio) is used extensively today by both academic and industrial institutions for the same reasons as highlighted by Streisinger in 1981 when studying vertebrate development.21 The generation time is only 3-4 months; at weekly intervals mature female [zebrafish] lay several hundred eggs which develop rapidly and synchronously outside the mother; the fish are small [approximately 3 cm when mature], hardy and easy to care for. Large scale screening of mutants is possible because free-swimming 7-dayold fish exhibit many behavioural and morphological traits of the parents but are only a few millimetres long.21,

22

These traits together with their transparency, availability and ease of

formation of transgenic animals, as well as their low chemical requirement for testing have endeared the species to developmental biologists. The breadth and depth of research applications utilizing the zebrafish model is highly diverse and ranges from ototoxicity to oncology and plastic surgery (cranial suture) to population effects.23-26 10 ACS Paragon Plus Environment

Genomic comparisons have

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demonstrated that 71.4% of human genes have an orthologue in the zebrafish, and of the potential human disease related genes (potential drug targets) this percentage increases to 82%.27 In addition, development of new techniques for genetic sequence alteration in particular, Transcription Activator- Like Effector Nucleases (TALENs) and more recently, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), have expanded research applications of zebrafish. These techniques allow for increasing specificity of mutation and tailoring of the fish to specific research goals and needs. A more detailed list of available techniques, which can be applied to the zebrafish, has been reviewed in 2014 by Lee et al.28, 29 Altogether, zebrafish has been described as a “convenient and predictive animal model [which] can serve as an intermediate step between cell-based evaluation and mammalian animal testing.30 3.1 Progress in Developing Zebrafish Screening Assays In recent years there has been substantial efforts in integrating the zebrafish embryo-larva model into screening assays for developmental toxicity.31 An early study on the use of zebrafish for teratogenicity testing of pharmaceutical agents evaluated 30 pharmaceutical compounds in dechorinated zebrafish embryos and yielded promising results with approximately 87% total concordance and < 15% error rate in misclassification of in vivo teratogens and non teratogens [Figure 3].32 Subsequent evaluation in other laboratories suggested the chorion may not be a significant barrier for compound exposure and by removing this step, would make the assay more conducive to automation. Using the basic framework of the dechorinated assay, a number of pharmaceutical and biotechnology companies formed a consortium to develop and evaluate a chorion-on developmental toxicology assay.

Approximately 60 compounds were evaluated in

multiple wild type strains by various laboratories to establish a harmonized protocol and rigorously assess predictivity. The harmonized zebrafish embryo developmental toxicity assay 11 ACS Paragon Plus Environment


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(ZEDTA) has been shown to have a predictive value of between 65-78% at the respective laboratories that conducted inter-laboratory assessment on both proprietary and non-proprietary compounds.33, 34 Ultimately, the harmonized protocol required multiple layers of screening, where compounds that were classified as negative for teratogenicity based upon the lack of achieving a concentration that achieved 25% lethality (LC25 ) and dysmorphology achieved at the highest concentration were subsequently evaluated for compound uptake and those with 75% concordance with existing in vivo models when tested with >10 compounds of known in vivo embryotoxicity potential.67 Other than the obvious ethical concerns of using human ESC, there are other disadvantages associated with developing humanized ES assays. For instance there is a very low number of known definitive human teratogens to be used in the validation of these tests. Animal data is readily available for a large number of compounds; therefore, concordance and predictivity values of animal-based in vitro systems will likely be more accurate and apply directly to the current in vivo animal test paradigm. Furthermore, demonstration of reproducible human ESC culture and optimized cell differentiation conditions is still necessary. The advantages and disadvantages of using mouse and human ESC are equally valid; advances will likely continue using human cells; however, until predictivity values are in place, the animal-based models will continue to be utilized. Perhaps in the future, both models will be used in unison or on a tiered testing strategy approach. An alternative to human ESC are human induced pluripotent stem (hIPS) cells; however, these cells have not been widely tested for embryotoxicity and teratogenicity testing. Currently there are some challenges that will need to be addressed with additional research to enable hIPS as potential replacements for human or mouse ESC for developmental toxicology screening. For example, recently, Gore et al. demonstrated an increase in nucleotide mutations associated with cell reprogramming in 22 hIPS cell lines. 68 Additionally, there are accumulating data to suggest 16 ACS Paragon Plus Environment

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there are vast differences between ESC and hIPS cells in terms of gene and microRNA expression.69-72 Additional research of these areas and potential some pilot evaluations comparing hIPS, hESC and mSC for predictivity in correctly classifying teratogens and non teratogens should provide further information on whether hIPS can potentially replace the current cell models.

5. Future Applications of Developmental Toxicology Testing in Industry: Several years ago, the National Research Council published a seminal report regarding future visions of toxicological testing Toxicity Testing in the 21st Century: Visions and Strategy (Tox21).5

The purpose of this report was to provide a vision to modernize toxicological

evaluation of compounds, moving away from animal models and replacing in vivo studies with a combination of in vitro assays and systems biology approaches. Altogether, this would result in substantial reduction of animal use across industry including pharmaceutical, chemical, agriculture-chemical and consumer products areas to find alternatives to whole animal testing. A driver towards finding new strategies in reducing animal use in toxicology work was in response to impending European REACH (Registration, Evaluation, Authorization and restriction of Chemicals) legislation, which requires toxicological assessment of all chemicals sold in Europe in quantities of more than 1 ton/per year. Standard toxicological assessment is not feasible since >68,000 compounds will require testing, and the task is required to be complete within the next decade. Furthermore, about 70% of the animals used in such toxicological assessments are related to reproductive and developmental toxicology tests.1, 73



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The three developmental models described in this review have promising utility to potentially reduce or replace whole animal tests but will need further evaluation. For instance, streamlining morphological endpoints and building prediction models for the zebrafish assays would increase practicality of that assay and make it more conducive to automation. Also, harvest and preparation of rodent embryos for culture requires specialized training due to the substantial skill involved in removing extraembryonic membranes, while maintaining integrity of the embryo. These steps will likely not be automated at least with current available technology. However, other parts of the WEC assay have automation potential, including gassing procedures and also imaging and scoring, especially in context of working with prediction models with only a few endpoints, such as the streamlined WEC assay.

Furthermore, complementary WEC

species such as rodent and rabbit may have potential to be combined in screening work to increase the predictivity. Further evaluation of existing statistical predication models with extended inclusion of broader classes of chemicals are required to determine applicability across much broader chemical space than solely pharmaceutical compounds. Such efforts may potentially lead to readjusting and optimizing the prediction models to suit broader chemical space or alternately lead to specific assay designs, endpoints and statistical models that best fit the chemistry for accurately predicting teratogenic outcome. Furthermore, test concentrations that more accurately represent maternal exposure for fetal adverse effect and no observed adverse effect level in vivo will improve ability to obtain risk assessment information from in vitro systems. Finally in vitro developmental toxicity data could serve broader applications such as computational chemistry, which may expand understanding of a compounds’ Structure Activity Relationship (SAR), drug penetration /concentration within the embryo-larvae and teratogenic signals.


As a SAR-

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teratogenicity database grows, eventually algorithms may be developed to predict teratogenic liability of small molecules by in silico approaches. Ultimately, an integrated or tiered strategy using WEC, zebrafish embryo culture and mouse embryonic stem cell culture assays (or incorporation of other developmental assays) are envisioned to improve predictivity of teratogenic potential. Through collaborative projects such as the ZEDTA consortium and cross-pharmaceutical discussions with the FDA and ICH, insight has been gained into how multiple assays have been implemented within the pharmaceutical industry. What is clear is that there is no single approach. Some companies tend to use the rat WEC as the standard precursor to a full mammalian developmental toxicity study and most have something in place prior to this to provide an early warning ‘flag’ at the lead selection-lead optimisation stage of the new drug development pipeline, or to prioritize future mammalian testing regimes. Outside of serving as a stand-alone assay, some laboratories integrate the zebrafish assay results with general toxicology data, chemical background history and/or in silico assessments for a weight of evidence approach for teratogenic classification or sometimes it is used within a tiered assessment integrated with other developmental toxicology assays (Stedman and Augustine-Rauch, personal communications).74

Some labs have begun multi-assay

performance testing and have reported enhanced predictivity by using multiple developmental toxicology assay datasets (Augustine-Rauch et al, in press).74 Multiple assay approaches using tiered screening or frequency testing (i.e. final teratogenic classification of a compound made from most frequent classification outcome of the respective group of developmental toxicology assays) has resulted in 88-89% predictivity (Augustine-Rauch et al in press). Additionally, regulatory agencies have initiated technology reviews with researchers in this area to learn more about the state of the science. It is anticipated that the review of the Detection of Toxicity to 19 ACS Paragon Plus Environment


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Reproduction for Medicinal Products & Toxicity to Male Fertility guideline (detection of toxicity to reproduction for medical products and toxicity to male fertility) may incorporate alternative models.75

Obtaining rigorous performance assessment and protocol harmonization as was

achieved with ZEDTA brings the effort one step closer to understanding potential and gaps of this assay and likely similar efforts will follow to harmonize and rigorously test other developmental toxicology assays that may be tiered or run together or the use of a weight of evidence approach to enhance overall predictivity to teratogenic liability.


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Acknowledgements: We are grateful to all of our colleagues who have participated in studies supporting the developmental toxicology assays described in this review.



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Abbreviations: Tox21: reference to National Research Council 2007 report on future visions of toxicological testing (Toxicity Testing in the 21st Century: Visions and Strategy ) REACH: Registration, Evaluation, Authorization and restriction of Chemicals ECVAM: European Center for the Validation of Alternative Methods ICH: International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use WEC: Whole Embryo Culture IC50: Concentration that causes 50% inhibition of an endpoint being measured NIH3T3: mouse embryonic fibroblast cell line DMS: Dysmorphology Score Cmax: Maximum systemic concentration achieved by a compound when dosed in vivo TALENs: Transcription Activator- Like Effector Nucleases CRISPRs: Clustered Regularly Interspaced Short Palindromic Repeats ZEDTA: Zebrafish Embryo Developmental Toxicity Assay LC25: concentration that achieved 25% lethality µM: micromolar concentration P450: Cytochrome P450 enzyme system CYP: Cytochrome P450 enzymes DarT: zebrafish assay that combines the use of a metabolic activation system using mammalian hepatic microsomes and zebrafish embryo culture ES cell: Embryonic Stem ESC: Embryonic Stem Cell EST: Embryonic Stem cell Test ES D3: Line of murine embryonic stem cells used in the EST ID50: The compound concentration that causes 50% cell differentiation inhibition RT-PCR: Reverse Transcriptase- Polymerase Chain Reaction 22 ACS Paragon Plus Environment

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MESCA: Molecular Embryonic Stem Cell Assay miRNA: micro Ribo Nucleic Acid ESNATS: Embryonic Stem Cell Based Novel Alternative Testing Strategies hIPS: human induced pluripotent stem hESC: human Embryonic Stem Cell mSC: mouse Stem Cell SAR: Structure Activity Relationship FDA: Food and Drug Administration



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Author Biographies:

Cindy Zhang received her BS in Wuhan, China and M.S. in Aquatic Biology at the Asian Institute of Technology in Bangkok, Thailand. She received a second M.S. in Immunology and Molecular Pathology from University of Florida. She worked in the Center for Cancer Treatment and Research in Richland Memorial Hospital affiliated with University of South Carolina and at GlaxoSmithKline and Bristol Myers Squibb Co. (BMS) in toxicology research. In recent years she has engaged in establishment of predictive models for supporting the rat and zebrafish in vitro teratogenicity screen assays.

Jonathan Ball received his BSc in Biochemistry and PhD. from the University of Wales and University of Exeter (UK), respectively. He joined AstraZeneca as the technical lead for nonmammalian alternatives for the assessment of Developmental Toxicology, which was relocated to Exeter University in 2014. His research focuses continue on evaluating applications of the zebrafish as a toxicity model for both industrial and academic research. 31 ACS Paragon Plus Environment


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Julie Panzica-Kelly obtained a B.S. in biology from Pennsylvania State University. She joined Bristol Myers Squibb where she has been engaged in novel developmental toxicology assay and statistical model development including the zebrafish embryo culture assay and molecular embryonic stem cell assay (MESCA). She completed a Masters' degree in developmental biology at Thomas Jefferson University in Philadelphia, Pennsylvania. Her research interests include molecular pathways of development and how they relate to mechanisms of teratogenicity. Julie is an associate member of the Society of Toxicology and a Member of the Mid Atlantic Teratology Society.

Karen Augustine-Rauch received her BS in biology from Stockton University and Ph.D. in Cell Biology and Anatomy at the University of North Carolina. She was a post-doctoral fellow in Discovery in the Experimental Hematology Group at Amgen. She joined GSK and headed the Molecular Teratology group and later joined BMS as the Associate Director of Reproductive Toxicology. She is currently a Research Fellow in Discovery Toxicology. Her recent research focuses on approaches to proactively assess teratogenicity profiles of discovery compounds and


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studies associated with evaluating mechanisms of teratogenicity. She is a member of the Teratology Society and Society of Toxicology.



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Table 1. Morphological Score System Scores

Morphology Description

5

Structure is entirely normal for developmental stage

4

Subtle variation in morphology, recoverable developmental delay or anomaly

3

Structure has one mild abnormality

2

Moderate malformations with 2 or more abnormalities

1

Severe malformations with multiple abnormalities

0.5

Structure is not evident by gross morphology

0.1

default score to represent embryo lethality

Reprinted from Zhang, C.X., Danberry, T., Jacobs, M.A., Augustine-Rauch, K. (2010) A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture. Birth Defects Res., Part B 89, 485-492,76 with permission from John Wiley and Sons.


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Figure Legends: Figure 1: Rat embryo developmental stage for whole embryo culture. A. Extra-embryonic structures and morphology of GD9 rat embryos at the beginning culture. Rat embryos at this stage are very primitive and are still undergoing gastrulation (establishment of the three germ layers of the body plan) as well as initiating neurulation (formation of the primitive brain and spinal cord and establishment of the axis). B. GD11 rat embryos at the end of 44-48h of culture. Organogenesis is fully active at this stage with various craniofacial structures and organ systems established. Figure 1B was adapted from Augustine-Rauch, K., Zhang, C. X., and Panzica-Kelly, J. M. (2010) In vitro developmental toxicology assays: A review of the state of the science of rodent and zebrafish whole embryo culture and embryonic stem cell assays. Birth Defects Res., Part C 90, 87-98,10 with permission from John Wiley and Sons.

Figure 2: Representative score assignment for heart morphology. The integrity of the outflow tract and chambers are considered together when assigning a score for the heart. The respective score examples represent scores assigned if only that particular structure was affected. The scores would be reduced by at least 1if additional structures were affected on the other side of the heart, depending upon severity of the abnormality. A. Left side of a heart with normal ventricle and atrium. B. Ventricle slightly small but within normal range. C. Delineation between ventricle and atrium chambers is not clear. An abnormal dark / dense tissue in the ventricle chamber (arrow). D. Both ventricle and atrium chambers have blood congestion and are small. An enlarged and translucent outflow tract is observed above the chambers (arrow). E. Small chambers with abnormal orientation and a very short outflow tract (arrow). F. Right side of a heart with normal “U” shaped outflow tract (dotted line). G. Outflow 35 ACS Paragon Plus Environment


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tract slightly narrowed (arrows). H. A kink formed in outflow tract (dotted line). I. Outflow tract very short and narrow with a kink (dotted line). J. Abnormally shaped and shortened outflow tract (dotted line). Reprinted from Zhang, C.X., Danberry, T., Jacobs, M.A., Augustine-Rauch, K. (2010) A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture. Birth Defects Res., Part B 89, 485-492,76 with permission from John Wiley and Sons.

Figure 3. Diagram of the dechorinated zebrafish developmental toxicology assay. Fertilized eggs were collected at approximately 4 hours post fertilization and chorions removed by enzymatic treatment and manual dissection. Compounds were administered to gastrulation-stage embryos (equivalent staging as post-implantation mammalian embryos receiving 1st dose in in vivo teratology assessments). The embryos were cultured for 5 days and evaluated for viability, growth and developmental abnormalities. Reprinted from Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., and Augustine-Rauch, K. A. (2010) Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res., Part B 89, 66-77,32 with permission from John Wiley and Sons.

Figure 4: Embryonic Stem Cell (EST) method schematic. Cells are cultured in hanging drop cultures for 3 days and then grown in suspension for an additional 2 days. On day 5, individual cell clusters are transferred to 24-well plates and cultured for an additional 5 days. Cardiomyocyte beating evaluation is performed on day 10 of the assay. Reprinted from Reproductive Toxicology, 27, van Dartel, D. A., Pennings, J. L., Hendriksen, P. J., van Schooten, 36 ACS Paragon Plus Environment

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F. J., and Piersma, A. H., “Early gene expression changes during embryonic stem cell differentiation into cardiomyocytes and their modulation by monobutyl phthalate,” 93-102, 2009, with permission from Elsevier.52



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Figures: Figure 1.


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Figure 2.



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Figure 3.


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Figure 4.



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An Overview of the Steps Toward Progressing In Vitro Developmental Models For Teratogenic Assessment of Chemicals 216x121mm (96 x 96 DPI)

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In Vitro Developmental Toxicology Screens: A Report on the Progress

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