HET-CAM test (hen’s egg-chorioallantoic membrane test) is a test used to determine the irritation potential of substances and is an alternative test to the Draize Rabbit Eye Test. In the test, incubated hen’s eggs are opened carefully on day 9 and the chorioallantoic membrane (CAM) is exposed.

Test substances, pure or diluted solutions, are placed directly on the exposed CAM membrane (Figure 1), and the membrane is inspected visually through a microscope. Time for reaction to occur on the membrane is recorded during a 5 min period. The reaction outcomes are haemorrhage (Figure 2), coagulation and lysis. Based on the reaction time, an irritation score, IS, (Kalweit et al., Toxicol in vitro 1990;4:702–704) is calculated and the substances are classified on the basis of the value of the score. Test solutions scoring 0–0.9 are classified as nonirritative, scores 1–4.9 as slightly irritative, scores 5–8.9 as moderately irritative and scores 9–21 as strongly irritative.

NIOM has used the test method to evaluate different substances used in dentistry such as extracts from different dental restorative materials, dental adhesives and primers, substances in toothpaste and hydrofluoric acid (HF). The HET-CAM test showed that a HF concentration of 0.05% was slightly irritative, 0.10% moderately irritative and 0.20–1% strongly irritative. In dentistry, HF is mainly used as a component for etching of ceramics and if spilled on soft tissue it can give severe injuries. In a recent study, the HET-CAM test showed that HF combined with potassium fluoride (KF) to potassium hydrogen difluoride (KF·HF) reduces the irritation potential considerably.

HET-CAM testHET-CAM test





Figure 1: CAM membrane before exposure.
Figure 2: CAM membrane exposed to substance that gives haemorrhage.


NIOM Newsletter October 2013