Validation, Optimization, and Application of the Zebrafish Developmental Toxicity Assay for Pharmaceuticals Under the ICH S5(R3) Guideline.

Yi-Sheng Song,Cheng-Da Zhang,Feng Xie,Chun-Qi Li,Jing Liu,Yan-Feng Huang,Yi Peng,Ming-Zhu Dai,Li-Jiang Zhang,Yong Zhang,Chen-Xia Zhu

doi:10.3389/fcell.2021.721130

Abstract

The zebrafish as an alternative animal model for developmental toxicity testing has been extensively investigated, but its assay protocol was not harmonized yet. This study has validated and optimized the zebrafish developmental toxicity assay previously reported by multiple inter-laboratory studies in the United States and Europe. In this study, using this classical protocol, of 31 ICH-positive compounds, 23 compounds (74.2%) were teratogenic in zebrafish, five had false-negative results, and three were neither teratogenic nor non-teratogenic according to the protocol standard; of 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish and two had false-positive results. After we added an additional TI value in the zebrafish treated with testing compounds at 2 dpf along with the original 5 dpf, proposed a new category as the uncategorized compounds for those TI values smaller than the cutoff both at 2 dpf and 5 dpf but inducing toxic phenotypes, refined the testing concentration ranges, and optimized the TI cut-off value from ≥ 10 to ≥ 3 for compounds with refined testing concentrations, this optimized zebrafish developmental assay reached 90.3% sensitivity (28/31 positive compounds were teratogenic in zebrafish) and 88.9% (40/45) overall predictability. Our results from this study strongly support the use of zebrafish as an alternative in vivo method for screening and assessing the teratogenicity of candidate drugs for regulatory acceptance.

Highlights

Developmental toxicity and teratogenicity represent a severe safety problem that causes approximately 5–10% of the congenital abnormalities of human newborns by teratogenic agents (Seiler et al, 2009)
The results derived from this study indicated that the zebrafish developmental toxicity assay optimized and validated in this report is a reliable and reproducible non-mammalian in vivo method for screening and assessing the teratogenicity
Developmental toxicity of 45 International Conference of Harmonization (ICH) categorized positive or negative compounds was assessed at 2 dpf to 5 dpf zebrafish using parameters presented in Tables 3, 4, and Supplementary Tables 2–4, and LC25, no observed adverse effect level (NOAEL), and teratogenic index (TI) were calculated as described in Materials and Methods

Summary

Introduction

Developmental toxicity and teratogenicity represent a severe safety problem that causes approximately 5–10% of the congenital abnormalities of human newborns by teratogenic agents (Seiler et al, 2009). It was estimated that 5816 traditional testing animals and a £1,883,200 cost would be required for the assessment of the developmental toxicity of one chemical according to the Organization for Economic Co-operation and Development (OECD) guidelines TG 414 (Organization for Economic Co-operation, and Development [OECD], 2001a), TG 416 (Organization for Economic Co-operation, and Development [OECD], 2001b), TG (Organization for Economic Cooperation and Development [OECD], 1995), and TG (Organization for Economic Co-operation and Development [OECD], 1996), no matter how time-consuming (Fleischer, 2007) This situation has urged research into alternative methods for developmental toxicity testing, namely, the whole embryo culture (WEC) test (Webster et al, 1997), the mammalian micromass (MM) test (Flint, 1993), and the embryonic stem cell test (EST; Spielmann et al, 1997). These tests do not cover the whole period of embryo development (Spielmann et al, 2006)

Methods

Results

Conclusion