HET-CAM bioassay in comparison to the croton oil test for investigating steroidal and non-steroidal compounds

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Adelheid H. Brandtner
Franz Quehenberger
Asima Chakraborty
Jutta Polligger
Silvio Sosa
Roberto Della Loggia


In this study the irritation phenomena at the chorioallantoic membrane of incubated hen's eggs as an in vitro model (HET-CAM assay) were investigated in comparison to the in vivo croton oil test by including hydrocortisone, indomethacin, phenylbutazone, acetylsalicylic acid, rutin, quercetin, apigenin, and p-coumaric acid as steroidal and non-steroidal test substances. For the first time the two methods were compared in a valid way with the perspective of a realistic reduction of animal experiments. It should be investigated whether an in vitro-in vivo correlation exists and, if there is any possibility, to replace the in vivo model by an in vitro test system.
Both bioassays were able to demonstrate the anti-inflammatory potency of the constituents tested. The determination of the anti-inflammatory activity of all compounds in the two test systems showed individual trends of inhibitory effects.
However, the in vitro HET-CAM test was much more sensitive in comparison to the in vivo croton oil test. The croton oil test gave dose-effect correlations in the anti-inflammatory substances investigated. The modified HET-CAM assay did not provide clear dose-effect ratios. The HET-CAM assay is an inexpensive test being easy to manage after a short practical training. Because of its sensitivity the HET-CAM assay could be considered a suitable tool for qualitative testing of the anti-inflammatory activity of substances if no appropriate dose-effect curves are required. From these results it can be concluded that the different courses of the dose-effect curves may be primarily due to different mechanisms of action.

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Brandtner, A. H., Quehenberger, F., Chakraborty, A., Polligger, J., Sosa, S. and Della Loggia, R. (2002) “HET-CAM bioassay in comparison to the croton oil test for investigating steroidal and non-steroidal compounds”, ALTEX - Alternatives to animal experimentation, 19(2), pp. 51–56. Available at: https://altex.org/index.php/altex/article/view/1102 (Accessed: 31 January 2023).