The red blood cell phototoxicity test (photohaemolysis and haemoglobin oxidation): EU/COLIPA validation programme on phototoxicity (phase II)

In the EU/COLIPA validation programme on "Photoirritation in vitro", two co re tests and a number of mechanistically based tests were carried out to ex amine their suitability as regulatory tests for phototoxicity testing. In t he 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 (Ph oto-RBC test), has passed through a prevalidation process during this progr amme. This test protocol combines two endpoints, photohaemolysis and met-ha emoglobin (met-Hb) formation. These endpoints are determined by measuring c hanges in the optical density of the haemoglobin spectrum at 525nm and 630n m, respectively. In addition. a prediction model was inserted into the Stan dard Operating Procedure (SOP) with two cut-off values: a photohaemolysis F actor (PHF) greater than or equal to 3.0 for photohaemolysis, and a Delta O Dmax greater than or equal to 0.05 for met-Hb formation. Three laboratories agreed to implement the SOP and to perform the study by testing 30 selecte d test chemicals 125 phototoxicants and 5 non-phototoxic chemicals,. The ou tcome of the study presents a good overall fit. including acceptable accura cy, sensitivity, and positive predictivity. The specificity and the negativ e predictivity are comparably low, due to the low number of non-phototoxic substances among the test chemicals. Further analysis of the data showed th at the transfer of the SOP from between laboratories could have been more e fficient, The results, especially of the lead laboratory, clearly indicate that an experienced laboratory can handle the SOP with high predictivity fu r phototoxicants and non-phototoxic substances. Finally, it was concluded t hat 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 .