Optimization and performance assessment of the chorion-off[Dechorinated] Zebrafish Developmental toxicity assay

Abstract

The Dechorinated Zebrafish Embryo Developmental toxicity assay was originally developed from a training set of 31 compounds and reported to be 87% concordant with in vivo teratogenicity data (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. 89, 66-77.). The assay includes scoring larva treated in a concentration range for malformations of specific morphological structures/organ systems. The model includes identifying a no-adverse-effect-level (NOAEL) and the concentration resulting in 25% lethality (LC25) at 5 days postfertilization. An LC25/NOAEL ratio ≥10 classifies a compound positive for teratogenic potential. A consortium effort evaluated a modified version of this assay which involved enzymatic chorion treatment instead of manual dissection and used experimental replicates for final classification. The modified assay achieved an 85% overall predictivity (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). Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay - progress report on phase I. Reprod. Toxicol. 33, 155-164.). The objective of this study was to perform a thorough performance evaluation of the dechorinated assay by repeating the original training set and testing additional compounds in experimental replicates. When the initial training set was repeated with inclusion of experimental replicates, the overall predictivity was 83%. Model performance was tested with an additional 34 compounds and achieved overall predictivity of 74%. When the training and test sets were combined (63 compounds) the assay's final sensitivity was 83% and the specificity was 71%. Total predictivity was 78% with relatively balanced predictivity for nonteratogens (77%) and teratogens (78%). The chorion-offassay achieved superior sensitivity (83%) compared with sensitivity (63-74%) reported by consortium efforts testing a similar assay with chorion-intact embryos (Ball, J. S., Stedman, D. B., Hillegass, J. M., Zhang, C. X., Panzica-Kelly, J., Coburn, A., Enright, B. P., Tornesi, B., Amouzadeh, H. R., Hetheridge, M., et al. (2014). Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay. Toxicol. Sci. 139, 210-219.). Additional protocol modifications were made to increase assay throughput.