Test No. 432: In Vitro 3T3 NRU Phototoxicity Test

         This Test Guideline describes a method to evaluate photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light.          Balb/c 3T3 cells are maintained in culture for 24 h for formation of monolayers. Two 96-well plates are pre-incubated with eight different concentrations of the test substance for 1 h. Thereafter one of the two plates is exposed to the highest non-cytotoxic irradiation dose whereas the other plate is kept in the dark. Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the Vital dye Neutral Red (NR) when measured 24 hours after treatment with the test chemical and irradiation. NR penetrates cell membranes by non-diffusion, accumulating in lysosomes. Alterations of the cell surface of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. Such changes result in a decreased uptake and binding of NR. It is thus possible to distinguish between viable, damaged or dead cells. To predict the phototoxic potential, the concentration responses obtained in the presence and in the absence of irradiation are compared, usually at the IC50 level, i.e., the concentration reducing cell viability to 50 % compared to the untreated controls.          Software to be used with TG 425, 432, 455. Click here.  Software not part of the Mutual Acceptance of Data.      