ReproTracker assay plates

Biology matters! Early teratogenicity assessments using ReproTracker

Case Study


Biology matters! Early teratogenicity assessments using ReproTracker

ReproTracker devtox



We made comparisons between ReproTracker and three in vitro teratogenicity high throughput assays, TeraTox, DevTox GLR, and hPST that have shorter assay durations.


By covering different germ layers and differentiated cell types, ReproTracker presented a more biologically advanced profile than other assays. It also presented higher accuracy in identifying teratogenicity than TeraTox and DevTox GLR.


In vitro teratogenicity assessments & ReproTracker

Of all the endpoints evaluated in a safety assessment, developmental and reproductive toxicity (DART) is one of the most challenging when developing alternative in vitro models. This means that companies must predict this endpoint using gold standard in vivo animal studies – despite the universal goal to adopt a more 3Rs-friendly strategy. Moreover, during the early stages of chemical selection and development, researchers rely on higher throughput in vitro assays to quickly and cost-effectively deselect potentially harmful chemicals. The pressure to develop predictive in vitro assays is even more urgent for the cosmetics industry which must tackle all toxicity endpoints without the use of animals. Despite this demand, there is still a lack of suitable in vitro alternatives to animal DART studies.

A particular challenge in predicting DART is the complexity of biological pathways involved in embryonic development. Moreover, there is currently no stand-alone single in vitro assay which can identify all teratogens. There are a limited number of in vitro assays which aim to detect potential teratogens, each with advantages, such as higher throughput or ease of endpoint measurement. Several assays use directed differentiation of stem cells to assess the effect of chemical exposure on metabolite or protein markers and/or expression of the early germ-layer genes as a surrogate for teratogenicity assessment. These include the human pluripotent stem cell test (hPST), the TeraTox and the DevTox Germ Layer Reporter Platform (DevTox GLR), all of which are 2- to 7-day high throughput screening (HTS) assays.

ReproTracker in hood

One aim of HTS assays is that they are of a short duration and possible to complete within the working week. However, is this saving on assay duration at the cost of the overall accuracy of the assay? To answer this question, we compared three in vitro teratogenicity HTS assays with the more biologically advanced ReproTracker (Table 1). ReproTracker follows the differentiation of hiPSCs into three germ layer cell lineages, mesoderm, endoderm and ectoderm, leading to the development of cardiomyocytes, hepatocytes and neuronal cells, respectively (Figure 1). This means that the duration of the assay is longer than others (up to 21 days) but, crucially, it recapitulates the lineage-specific pathways during early embryonic development. By extending the duration of the assay, the effects of the test chemical on the morphology and function of the differentiated cells are monitored, along with changes in expression of biomarker genes.

ReproTracker assay plates

Table 1. Overview of the assays compared.

AssayDuration (days)Germ layers coveredTissue specific differentiationEndpoints





Neural cells

6 biomarkers for early and late-stage differentiation: FOXA2, AFP (liver); BMP4, MYH6 (heart); PAX6, Nestin (neural cells)

Functionality and morphology assessment

A LOAEL is also derived from the assay




NoneExpression of 87 early developmental markers (germ layer genes)
DevTox GLR2Mesoderm



NonePercentage of SOX17-, SOX2-, or BRA-positive cells
hPST3Mesoderm onlyNoneEarly lineage endoderm marker SOX17 (+ larger panel gene expression profiling)

Based on whether the IC50 of SOX17 down-regulation is higher or lower than a threshold of 30 µM


Figure 1. Schematic representation of hiPSCs differentiation towards cardiomyocytes, hepatocytes and neural rosettes in ReproTracker.

ReproTracker differentiation figure

A Case Study Comparing ReproTracker with TeraTox, DevTox GLR, and hPST

Our Approach

So, does this larger biological coverage give ReproTracker an advantage over other shorter duration human-based assays regarding its predictive of teratogenic potential? To make the most relevant comparison, the assays should be compared using the same chemical sets. Therefore, we collected teratogenicity outcomes for a set of well-known in vivo teratogens and non-teratogens tested in the hPST, TeraTox, DevTox GLR and ReproTracker, and then made pairwise comparisons of assays using common chemicals.

Higher Predictivity

Based on a common set of 21 chemicals, ReproTracker correctly differentiated between all teratogens and non-teratogens (i.e. the accuracy was 100%), whereas the accuracy of the TeraTox assay was only 76%. Similarly, based on 23 compounds, the accuracy of ReproTracker was higher (96%) than the DevTox GLR assay (83%). When a small chemical set of only 11 chemicals was compared, the accuracy of the hPST and ReproTracker were 100% and 82%, respectively. While this indicates a superiority of the hPST, this assay requires a threshold concentration of 30 µM to achieve this predictivity. This means it cannot be used for hazard identification screening if lower concentrations are tested e.g. due to poor solubility.

It is likely that the higher predictivity of the ReproTracker compared to other in vitro assays can be attributed to its ability to track a much wider spectrum of cellular processes involved in early embryonic development. In contrast to ReproTracker, other assays fail to detect chemicals affecting differentiated tissue-specific markers occurring between 3-21 days. Thus, unlike the shorter stem cell-based assays, ReproTracker can also provide insight into the mechanism of teratogenicity based on tissue-specific markers, which is important when designing follow-up investigations. For example, Dasatinib was observed to affect the cardiac biomarker in ReproTracker, whereas Artesunate and Imatinib altered liver biomarkers. An interesting example of a chemical which is not picked up as a teratogen by the TeraTox assay – but is a clear in vivo teratogen and positive in ReproTracker – is warfarin. In ReproTracker, warfarin affected the tissue-specific markers (AFP or MYH6), underscoring the importance of biological coverage in detecting a wider range of potential teratogens.

ReproTracker imaging


For those wishing to establish a strategy for evaluating DART earlier in development, in vitro assays are clearly the way to go. Obviously, each in vitro model has its advantages and disadvantages, and a combination of these is needed to be able to select candidates with the best safety profile. While 2- to 3-day HTS assays can provide an initial assessment, ReproTracker provides an accurate prediction of in vitro teratogenicity by capturing the complex biology of embryogenesis and enabling an indication of the organs affected. When it comes to DART assessments, biology matters!

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Poster - Capturing complex biology is critical for accurate in vitro prediction of developmental toxicity - BDRP 2024 small

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Jaklin M, Zhang JD, Schäfer N, Clemann N, Barrow P, Küng E, Sach-Peltason L, McGinnis C, Leist M, Kustermann S. Optimization of the TeraTox Assay for Preclinical Teratogenicity Assessment. Toxicol Sci. 2022 Jun 28;188(1):17-33. doi: 10.1093/toxsci/kfac046. PMID: 35485993; PMCID: PMC9237991.

Jamalpoor A, Hartvelt S, Dimopoulou M, Zwetsloot T, Brandsma I, Racz PI, Osterlund T, Hendriks G. A novel human stem cell-based biomarker assay for in vitro assessment of developmental toxicity. Birth Defects Res. 2022 Nov 15;114(19):1210-1228. doi: 10.1002/bdr2.2001. Epub 2022 Mar 14. PMID: 35289129

Gamble JT, Hopperstad K, Deisenroth C. The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test. Toxics. 2022 Jul 13;10(7):392. doi: 10.3390/toxics10070392. PMID: 35878297; PMCID: PMC9321663.

Kameoka S, Babiarz J, Kolaja K, Chiao E. A high-throughput screen for teratogens using human pluripotent stem cells. Toxicol Sci. 2014 Jan;137(1):76-90. doi: 10.1093/toxsci/kft239. Epub 2013 Oct 23. PMID: 24154490.

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