184 • Alternatives to Animal Use in Research, Testing, and Education 
Figure 8-1.— Chronological Sequence of Chick 
Embryo Chorioallantoic Membrane Assay 
Day 0 
Day 17 
Day 0. Fertile eggs are incubated at 37° C. Day 3. The shell is 
penetrated in two places: A window is cut at the top, and 1.5 to 2 
milliliters of albumin is removed with a needle and discarded. The 
chorioallantoic membrance forms on the floor of the air space, on 
top of the embryo. The window is taped. Day 14. A test sample is 
placed on the embryonic membrane and contained within a plastic 
ring. Day 17. The chorioallantoic membrane is evaluated for its 
response to the test substance, and the embryo is discarded. 
SOURCE: J. Leighton, J. Nassauer, and R. Tchao, “The Chick Embryo in Toxicol- 
ogy: An Alternative to the Rabbit Eye,” Food Chem. Toxicol. 23:293-298. 
Copyright 1985, Pergamon Press, Ltd. 
laries, and veins, and is technically easy to study. 
An embryonic membrane tested after 14 days of 
incubation responds to injury with a complete in- 
flammatory reaction, a process similar to that in- 
duced in the conjunctival tissue of the rabbit eye. 
The embryonic membrane can show a variety of 
signs of irritation and has capabilities for recov- 
ery (59,60). 
Assessment of toxicity is made and the embryo 
is discarded on about day 17 of incubation. The 
criteria used for macroscopic rating of lesions on 
the chorioallantoic membrane are listed below (59): 
• size, 
• contours and surface, 
• color, 
• retraction of surrounding chorioallantoic 
membrane, 
• spokewheel pattern of vessels, 
• overall grade of severity, and 
• necrosis (confirmed microscopically). 
Although this is, strictly speaking, an in vivo test, 
the chorioallantoic membrane does not have nerve 
cells, and thus it is unlikely that the organism ex- 
periences any discomfort. In addition, fertile eggs 
are inexpensive and do not require elaborate ani- 
mal room facilities. 
REPEATED-DOSE TOXICITY TESTS 
Repeated-dose toxicity testing involves the re- 
peated application of a substance to a biological 
assay system and subsequent measurement of 
many different effects of the substance. In re- 
peated-dose testing, the long-term effects of 
repeated, sublethal exposure to a substance are 
of interest, rather than acute, lethal effects. Cell 
cultures may be useful adjuncts for suspected tar- 
