Table 3.

Overview of experiments in zebrafish embryos.

Experiment	End point	Study design	Purpose
a.	General toxicity (developmental delay, malformations, lethality)	ZFET: single compounds in separate studies	Dose–range finding; estimate highest applicable concentration for exp. 2 and 3
b.	M-PQ angle	Single compounds in separate studies	Confirm predicted craniofacial malformation
			Establish (relative) potencies
			Determine NOAEL (used in exp. 3), ${\text{CED}}_{20}$ (used in exp. 4);
c.	M-PQ angle	Single compounds at NOAEL, compounds at NOAEL combined in a mixture and dilutions thereof	Confirm absence of effect at NOAEL
			Determine effect of combined compounds at NOAEL mixture and dilutions thereof
			Examine dose addition
d.	Marker gene expression	Single compounds in one study at one dose (${\text{CED}}_{20}$)	Confirm differential toxicological action

Note: ${\text{CED}}_{20}$ is modeled at the 20% effect level (compared with background), which was chosen to induce sufficient specific effect while avoiding non-target effects.²⁷ CED, critical effect dose; exp., experiment; M,-PQ, Meckel’s and palatoquadrate cartilages; NOAEL, no observed adverse effect level; ZFET, zebrafish embryo toxicity test.