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EC number: 200-568-1
CAS number: 63-91-2
The current ICCVAM recommended protocol for which HET-CAM is recommended
as a screening test to identify non-labelled ocular irritants uses an
analysis method (i.e., the Irritation Score IS(A)) which is based on
development of each of the three
HET-CAM endpoints at fixed time intervals of 0.5, 2, and 5 minutes
(Table 1; Lüpke 1985).
The numerical time-dependent scores for lysis, haemorrhage, and
coagulation (Table 2) are summed to give a single numerical value
indicating the irritation potential of the test substance on a scale
with a maximum value of 21.
Table 1: Scoring scheme
for irritation testing
Table 2: Classification
of cumulative scores
0 - 0.9
1 - 4.9
5 - 8.9
9 - 21
Criteria for an acceptable test
The HET-CAM assay is considered acceptable if the negative and positive
controls each induce a response that falls within the classification of
nonirritating and severely irritating, respectively. Historical control
studies indicate that using 0.9% NaCl, as a negative control, the IS
value was 0.0 (35 studies in the time period between 2010 and 2012).
Historical control studies demonstrate that using 1% SDS and 0.1 N NaOH
as positive controls results in IS values of approximately 8 to 12 or 17
to 19, respectively.
Lüpke, N. P., 1985: Hen's egg chorioallantoic membrane test for
irritation potential Fd. Chem. Toxic. Vol. 23, No. 2, pp. 287 - 291
ICCVAM. 2010: ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test -
Chorioallantoic Membrane (HET-CAM) Test Method. NIH Publication No:
10-7553, published 2010.
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