3T3 Neutral Red (NR) Assay
The 3T3 NR in vitro cytotoxicity assay procedure is used to determine the concentration of test article required to kill 20%, 50%, and 80% of NIH 3T3 cells in culture. These results are applied to a prediction model to estimate the in vivo LD50. Therefore, this test is a good nonanimal starting point to determine how toxic a test substance is.
NR is a weak cationic dye that readily diffuses through the plasma membrane and concentrates in lysosomes. Toxicants can alter the cell surface or the lysosomal membrane to cause lysosomal fragility and other adverse changes that gradually become irreversible. Such adverse changes cause cell death and/or inhibition of cell growth, which then decrease the amount of NR retained by the culture. Since the concentration of NR dye desorbed from the cultured cells is directly proportional to the number of living cells, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of NR after chemical exposure. The NRU assay uses a 96-well plate format to produce replicate measurements at eight test substance concentrations. The results are entered into a software package that predicts the inhibitory concentrations (ICs). The ICs are then applied to a prediction model to predict the in vivo lethal dose (LD50).
Lebrun Labs follows the procedure described in OECD 129.
Cosmetic and personal care product tests are available only through BioScreen Testing Services: http://www.bioscreen.com/index.php/forms-a-quotes