Use of Emerging Models for Developmental Toxicity Testing - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 39

Table 3.2 Comparison of Vertebrate Model Systems

Parameter Rat Rabbit Zebrafish

Ex utero gestation No No Yes

Embryogenesis 21 days 29 days 3 days

Embryos per mating 8-16 3-8 50-100

Transparent No No Yes

Microplate analysis No No Yes

Drug delivery Inject Inject Inject/solution

Cost per assay > $1000 > $1500 $0.10

Lethal effects are compared to controls to identify LC 50 , no observed effect
concentration (NOEC), and lowest observed effect concentration (LOEC) values.
The test uses a minimum of five concentrations and controls and 10 embryos per
concentration in two to three independent replicates. To assess lethality, after
treatment for 24 and 48 h, four end points are assessed: (1) coagulation of fertilized
eggs, (2) somite formation, (3) detachment of the tail bud from the yolk sac,
and (4) heartbeat.

3.4.2 Embryo Differentiation

Fertilized eggs undergo the first cleavage at 15min post fertilization and conse-

cutive synchronous cleavages form 4-, 8-, 16-, and 32-cell blastomeres. At these
stages, fertilized eggs can be clearly identified by development of a blastula.
Unfertilized eggs that do not undergo cleavage or eggs exhibiting obvious irregu-
larities during cleavage, such as vesicle formation and asymmetry, vesicle formation,
or defective chorions, are discarded.

3.4.3 Test Solutions

Test concentrations are prepared by diluting stock solution andmixing or agitating the
test substance in dilution water usually by mechanical means including stirring or
ultrasonification.

3.4.4 Exposure Time and Duration of Test

The test is initiated directly after egg fertilization and embryos are immersed in test
solutions before cleavage of the blastodisc begins. Approximately 40 eggs per
concentration are transferred to dilution water no later than 60min post fertilization.
Under a stereomicroscope (30

magnification), fertilized eggs are separated from
unfertilized eggs and then transferred to 24-well plates containing test solutions.
Then, at 120 min post fertilization, fertilized eggs are transferred to test chambers.