CrossDART

Background

Drugs intended for the treatment of women of child-bearing potential (WoCBP) must be tested for teratogenicity – the potential for the drug to cause harm to a developing embryo or fetus (1). Embryo-fetal development is a complex process which involves coordinated biological events that are susceptible to external influences. Exposure of the mother to a teratogenic drug can disrupt the normal formation of organs and may result in a wide range of malformations.

Studies to assess the potential teratogenicity of a drug typically involve evaluation in pregnant animals in two species (a rodent and non-rodent) as specified in the International Council for Harmonisation (ICH) S5 (R3) guideline (1). These embryo-fetal development (EFD) studies are generally conducted on the drug candidate once it has entered clinical development, but the animal models are not always predictive of humans and often do not provide mechanistic insight (2). Several alternative models exist that are used to predict teratogenicity during the selection of candidate drug molecules, but improved models are needed to address the current limitations and permit their broader use:

• In silico QSAR models have predictivity limited to defined chemical spaces (3, 4).
• Ex vivo embryo assay cultures (e.g. using rodent, rabbit) and non-mammalian models such as Caenorhabditis elegans or zebrafish may have limited mechanisms in common with human development (5-15).
• In vitro stem cell-based models (4, 16-29):
• Rely on adherent monolayer cultures or disorganised 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo.
• Can only recapitulate certain aspects of embryo development and do not cover mechanisms such as neurulation and the effect on the trophoectoderm/placenta.
• Do not provide an accurate method to extrapolate in vitro culture concentration to pharmacokinetic (PK) parameters of drug exposure in humans.
• Qualification of human in vitro models is hampered by the availability of human data and the limited relevance of animal data.  

There is a need to develop improved in vitro assays both in human and in preclinically relevant species (e.g. rat, rabbit, non-human primate (NHP)) to aid with translation and understanding of species-specific effects and human relevancy.

The Challenge

The aim of this Challenge is to develop and qualify an in vitro approach that can reliably predict early or surrogate indicators of teratogenicity of pharmaceutical drug candidates. The proposed approach should cover as many mechanisms of embryo development as possible. To support validation and translation, the approach must cover human as well as selected preclinical species used for EFD studies (rat, rabbit, NHP).

3Rs benefits

A standard EFD study requires multiple pregnant females (80 to100 for rodents and rabbits; and 48 for NHPs). In the case of EFD findings, additional mechanistic studies requiring further animals might be performed to identify if the findings are relevant to human. The packages of drugs approved by the FDA currently include around 67 EFD studies per year (around 6,000 animals) (30). For biopharmaceuticals which are not pharmacologically active in rodents, the EFD study can be replaced by an enhanced pre- and postnatal development (ePPND) study in NHPs.

This Challenge has the potential to deliver 3Rs benefits in the pharmaceutical industry by:

• Providing more predictive approaches that can be used for early screening and derisking to prevent drug candidates with teratogenic potential from progressing into animal studies.
• Use in mechanistic studies to investigate species differences/species-specific effects and human relevancy especially in the case of equivocal EFD results.
• Replacing in vivo studies for drugs which have a mode-of-action that is suspected to adversely influence morphogenesis, as specified in the ICH S5(R3) guideline.
• In the longer term, once qualified and accepted by regulatory authorities, substituting or deferring EFD studies in one of the two species required.

The assays developed through this Challenge will also have applicability to the food, chemical and agrochemical industries where teratogenicity assessment is also required.

References

1. ICH S5 (R3) guideline on reproductive toxicology: Detection of toxicity to reproduction for human pharmaceuticals - step 5 - Scientific guideline | European Medicines Agency (europa.eu)

    

2. Clements JM et al. (2020). Predicting the safety of medicines in pregnancy: A workshop report. Reprod Toxicol. 93: 199-210. doi: 10.1016/j.reprotox.2020.02.011
    

3. Cassano A et al. (2010). CAESAR models for developmental toxicity. Chem Cent J: Jul 29(4). doi: 10.1186/1752-153X-4-S1-S4

    

4. Wu S et al. (2013). Framework for identifying chemicals with structural features associated with the potential to act as developmental or reproductive toxicants. Chem Res Toxicol 26(12): 1840-61. doi: 10.1021/tx400226u

    

5. Genschow E et al. (2002). The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern Lab Anim 30(2): 151-76. doi: 10.1177/026119290203000204
    
6. Fort DJ and Mathis M (2018). Frog Embryo Teratogenesis Assay-Xenopus (FETAX): Use in Alternative Preclinical Safety Assessment. Cold Spring Harb Protoc 2018(8). doi: 10.1101/pdb.prot098319
    

7. Islas-Flores H et al. (2018). Evaluation of Teratogenicity of Pharmaceuticals Using FETAX. Methods Mol Biol. 1797: 299-307. doi: 10.1007/978-1-4939-7883-0_15

    

8. Brannen KC et al. (2010). Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res B Dev Reprod Toxicol. 89(1): 66-77. doi: 10.1002/bdrb.20223

    

9. Jarque S et al. (2020) Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates. Reprod Toxicol. 96: 337-48. doi: 10.1016/j.reprotox.2020.08.004

    

10. Tung EWY and Winn LM (2019). Mouse Whole Embryo Culture. Methods Mol Biol. 1965: 187-94. doi: 10.1007/978-1-4939-9182-2_13
     
11. Ozolinš TRS (2019). Rabbit Whole Embryo Culture. Methods Mol Biol. 1965: 219-233. doi: 10.1007/978-1-4939-9182-2_15
     

12. PREDART | Innovation Platform nc3rs.org.uk/crackit/predart

     

13. van der Voet M et al. (2021). Towards a reporting guideline for developmental and reproductive toxicology testing in C. elegans and other nematodes. Toxicol Res (Camb). 10(6): 1202-10. doi: 10.1093/toxres/tfab109
     
14. Bhalla D et al. (2023) DARTpaths, an in silico platform to investigate molecular mechanisms of compounds. Bioinformatics.  39(1): btac767. doi: 10.1093/bioinformatics/btac767
     
15. Cassar S et al. (2020) Use of Zebrafish in Drug Discovery Toxicology. Chem Res Toxicol. 33(1): 95-118. doi: 10.1021/acs.chemrestox.9b00335
     

16. Genschow E et al. (2000). Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr Mol Toxicol 13(1): 51-66. PMID: 10900407

     

17. Whitlow S et al. (2007) The embryonic stem cell test for the early selection of pharmaceutical compounds. ALTEX 24(1): 3-7. doi: 10.14573/altex.2007.1.3
     

18. Adler S et al. (2008). First steps in establishing a developmental toxicity test method based on human embryonic stem cells. Toxicol In Vitro 22(1): 200-11. doi: 10.1016/j.tiv.2007.07.013

     

19. Augustyniak J et al. (2019). Organoids are promising tools for species-specific in vitro toxicological studies. J Appl Toxicol. 39(12): 1610-1622. doi: 10.1002/jat.3815
     
20. Dreser N et al. (2020). Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances. Arch Toxicol 94(1): 151-171. doi: 10.1007/s00204-019-02612-5
     

21. Palmer JA et al. (2013). Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening. Birth Defects Res B Dev Reprod Toxicol 98(4): 343-63. doi: 10.1002/bdrb.21078

     

22. Palmer JA et al. (2017). A human induced pluripotent stem cell-based in vitro assay predicts developmental toxicity through a retinoic acid receptor-mediated pathway for a series of related retinoid analogues. Reprod Toxicol 73: 350-361. doi: 10.1016/j.reprotox.2017.07.011
     
23. Shinde V et al (2016). Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells. Stem Cell Res Ther 7(1): 190. doi: 10.1186/s13287-016-0449-2
     
24. Worley KE et al. (2018). Teratogen screening with human pluripotent stem cells. Integr Biol (Camb) 10(9):491-501. doi: 10.1039/c8ib00082d
     
25. Jamalpoor A et al. (2022). A novel human stem cell-based biomarker assay for in vitro assessment of developmental toxicity. Birth Defects Res 114(19):1210-1228. doi: 10.1002/bdr2.2001
     
26. Jaklin M et al. (2022). Optimization of the TeraTox Assay for Preclinical Teratogenicity Assessment. Toxicol Sci 188(1): 17-33. doi: 10.1093/toxsci/kfac046
     
27. Aikawa N et al. (2014). Detection of thalidomide embryotoxicity by in vitro embryotoxicity testing based on human iPS cells. J Pharmacol Sci 124(2): 201-7. doi: 10.1254/jphs.13162fp
     
28. Kanno S et al. (2022). Establishment of a developmental toxicity assay based on human iPSC reporter to detect FGF signal disruption. iScience 25(2): 103770. doi: 10.1016/j.isci.2022.103770
     
29. Mantziou V et al. (2021). In vitro teratogenicity testing using a 3D, embryo-like gastruloid system. Reprod Toxicol 105: 72-90. doi: 10.1016/j.reprotox.2021.08.003
     
30. Barrow P (2022). Review of embryo-fetal developmental toxicity studies performed for pharmaceuticals approved by FDA in 2020 and 2021. Reprod Toxicol. 112: 100-08. doi: 10.1016/j.reprotox.2022.06.012

Full Challenge information

Assessment information

Challenge Panel membership