Hen's Egg Test Chorioallantoic Membrane (HET-CAM)

Alternative Eye Irritation Testing

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Normal, vasculary structure of the Chorioallantoic Membrane - 10 day old egg
Normal Vascularity
Vasculary damage to the Chorioallantoic Membrane - 10 day old egg
Damaged Vascular structure

Draize Rabbit Eye Alternative

The HET-CAM is an Alternative to the Draize Rabbit Eye Test which mimics vascular changes in the chorioallantoic membrane, an analog for ocular conjuntiva, which can be used to determine the potential irritancy of a test substance. This method is based on that described in HET-CAM TEST, INVITTOX PROTOCOL Number 47 (1990), ISSN #0960-2194.


General Test System Parameters:

Fertile, 10 day old, White Leghorn eggs are used in the HET-CAM as an alternative to the Draize Rabbit Eye Test and is configured to accommodate 6 test groups - 3 test article concentrations, 2 positive controls and 1 vehicle control group.

Test System Justification:

The HET-CAM has been shown to be a qualitative method of assessing the potential irritancy of chemicals. The CAM is a complete tissue containing arteries, veins and capillaries, and is technically easy to study. It responds to injury with an inflammatory process similar to what one would observe in the conjunctival tissue of a rabbit's eye. Its well-developed vascularization provides an ideal model for ocular irritation studies.

Experimental Design:

Development: Upon receipt, the eggs will be placed in commercial incubators. On day 10 of development, the eggs will be removed from the incubator and candled to determine the viability of the embryo. A rectangular window will be removed from the shell directly over the air cell and the egg membrane will be carefully moistened with 2-3 ml 0.9% saline and returned to the incubator.

The eggs are then dosed and observed continuously for 5 minutes for the appearance of lysis, hemorrhaging and/or coagulation which is documented.

In addition, the eggs are scored for severity at 1 and 5 minutes. The severity of each reaction after 1 and 5 minutes is recorded.

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