Fishing for Teratogens: A Consortium Effort for a Harmonized Zebrafish Developmental Toxicology Assay

Abstract

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.

From cutting edge to guideline: A first step in harmonization of the zebrafish embryotoxicity test (ZET) by describing the most optimal test conditions and morphology scoring system

“…Van den Bulck et al [22] used a pre-determined 50 ng/larvae as maximum body burden, which was considered successful by Doshna et al [55] but showed to be too high for the tested compounds by Van den Bulck et al [22]. In addition, Ball et al [5] implemented the following strategy: higher dosing concentrations were employed with compounds which demonstrated no lethality, morphological malformations and low uptake (< 5%). So, they defined the minimum level of body burden as 5%.…”

“…Since 2009, a consortium of six biopharmaceutical companies are working hard to develop a harmonized ZET [2][3][4], however still technical differences remain and are the primary contributor to inter-laboratory differences in predictivity [5]. However, until now, no global harmonized experimental setup has been established.…”

Zebrafish in Toxicology and Environmental Health

“…The results of these toxicity tests were compared to mammalian data, and found to have an overall concordance of 60–70%. In a second phase of this consortium project, 38 proprietary pharmaceutical compounds were tested by two independent laboratories, and 79% of the classifications were the same between the laboratories, although the laboratories differed in their concordance with in vivo data (Ball et al, 2014). The Dechorionated Zebrafish Embryo Developmental toxicity assay was developed to identify the no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC 25 ) for a training set of 31 compounds (Brannen, Panzica-Kelly, Danberry, & Augustine-Rauch, 2010).…”

“…This approach yielded 87% concordance with published in vitro teratogenicity data (Brannen et al, 2010). Improvements to this assay, including enzymatic removal of the chorion, repeating the assay with a distinct set of text compounds, and using various zebrafish strains, have been attempted to make a direct comparison between the chorion-on data published by the pharmaceutical company consortium and the chorion-off data to determine whether the presence of the chorion affected the sensitivity and specificity of the zebrafish embryo assay (Ball et al, 2014; Brannen et al, 2010; Gustafson et al, 2012; Panzica-Kelly, Zhang, & Augustine-Rauch, 2015). …”

Zebrafish: A Marvel of High-Throughput Biology for 21st Century Toxicology

“…It stands to reason that economies of scale can be realized using the zebrafish in drug discovery. Consequently, a consortium of multinational pharmaceutical companies initiated the process of validating the zebrafish for use in their drug discovery pipeline (143). …”