Evaluation of the HET-CAM-TSA method as an alternative to the draize eye irritation test
L Gilleron1, S Coecke, M Sysmans
1Department of Genetic and In Vitro Toxicology, Janssen Research Foundation-BEERSE, Belgium.
Abstract:
The Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method was modified in our laboratory by means of microscopic evaluation, a clear description of the three in vitro endpoints (haemorrhage, lysis and coagulation) and the use of a test substance applicator (TSA). A previous study on 46 chemicals demonstrated the usage of the HET-CAM-TSA assay as a screening test for eye irritancy. In order to extend our database and to come to a more reliable conclusion concerning the use of the HET-CAM-TSA method, a second set of 60 test substances was tested. The in vitro irritation scores (IS) were compared with the in vivo modified maximum average scores (MMAS) calculated 24 hr after instillation. The MMAS irritancy threshold was set at 15.0. The results were analysed according to the Cooper's parameters (specificity, sensitivity and concordance with the Draize test) and the Pearson's correlation coefficient. It was concluded that the HET-CAM-TSA test was a valuable screening test. To compensate for the misclassifications generated, it was also concluded that the HET-CAM-TSA method should be considered as a part of a test battery, together with the Bovine Corneal Opacity-Permeability (BCOP) assay.
Insights
The modified Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) with a test substance applicator (TSA) effectively screens for eye irritancy. For improved accuracy, this in vitro method should complement other tests like the Bovine Corneal Opacity-Permeability (BCOP) assay.
Area of Science:
- Toxicology
- In vitro testing
- Ocular safety assessment
Background:
- The Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) assay is an established method for evaluating eye irritancy.
- Previous studies indicated the potential of the HET-CAM assay with a test substance applicator (TSA) as a screening tool for eye irritancy.
Purpose of the Study:
- To further validate and extend the database for the modified HET-CAM-TSA assay.
- To assess the reliability of the HET-CAM-TSA method for predicting in vivo eye irritancy.
Main Methods:
- Modification of the HET-CAM assay incorporating microscopic evaluation, defined endpoints (hemorrhage, lysis, coagulation), and a test substance applicator (TSA).
- Testing of 60 additional substances using the HET-CAM-TSA assay.
- Comparison of in vitro irritation scores (IS) with in vivo modified maximum average scores (MMAS) at 24 hours post-instillation.
Main Results:
- The HET-CAM-TSA test demonstrated value as a screening test for eye irritancy.
- Analysis using Cooper's parameters (specificity, sensitivity, concordance with Draize test) and Pearson's correlation coefficient was performed.
- Misclassifications were observed, highlighting the need for complementary testing.
Conclusions:
- The HET-CAM-TSA assay is a valuable screening tool for eye irritancy.
- To enhance accuracy and compensate for misclassifications, the HET-CAM-TSA method should be integrated into a test battery.
- The Bovine Corneal Opacity-Permeability (BCOP) assay is recommended as a complementary test within this battery.
