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The Red Blood Cell Phototoxicity Test (Photohaemolysis and Haemoglobin Oxidation): EU/COLIPA Validation Programme on Phototoxicity (Phase II)


Wolfgang J.W. Pape, Thomas Maurer, Uwe Pfannenbecker and Winfried Steiling

In the EU/COLIPA validation programme on “Photoirritation in vitro”, two core tests and a number of mechanistically based tests were carried out to examine their suitability as regulatory tests for phototoxicity testing. In the meantime, one core test, the 3T3 neutral red uptake phototoxicity test (NRU PT) has been validated and has been accepted by ECVAM and the European Commission. The second core test, the red blood cell phototoxicity test (Photo-RBC test), has passed through a prevalidation process during this programme. This test protocol combines two endpoints, photohaemolysis and met-haemoglobin (met-Hb) formation. These endpoints are determined by measuring changes in the optical density of the haemoglobin spectrum at 525nm and 630nm,  respectively. In addition, a prediction model was inserted into the Standard Operating Procedure (SOP) with two cut-off values: a photohaemolysis factor (PHF) ≥ 3.0 for photohaemolysis, and a ∆ODmax ≥ 0.05 for met-Hb formation. Three laboratories agreed to implement the SOP and to perform the study by testing 30 selected test chemicals (25 phototoxicants and 5 non-phototoxic chemicals). The outcome of the study presents a good overall fit, including acceptable accuracy, sensitivity, and positive predictivity. The specificity and the negative predictivity are comparably low, due to the low number of non-phototoxic substances among the test chemicals. Further analysis of the data showed that the transfer of the SOP from between laboratories could have been more efficient. The results, especially of the lead laboratory, clearly indicate that an experienced laboratory can handle the SOP with high predictivity for phototoxicants and non-phototoxic substances. Finally, it was concluded that the combined Photo-RBC test can be considered as a second in vitro test, which can be used advantageously to obtain some mechanistic information, in particular on photodynamic effects on cellular proteins and biomembranes.