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3T3 Neutral Red Uptake Phototoxicity Assay Applications

Test Material Compatibility

The 96-well plate based cytotoxicity assays using NRU are best suited for water-soluble materials; however, test materials may be dissolved in intermediate solvents so that they are compatible with the test system. At IIVS, a solubility test is performed to find the most suitable solvent when the solubility of a test chemical is unknown or unavailable.

As a Prediction of Systemic Toxicity

The Balb/c 3T3 Cytotoxicity Assay using a NRU viability endpoint can also be used to estimate starting doses for acute oral toxicity testing in rodents (the methods were initially proposed by ZEBET based upon the correlation of the in vitro cytotoxicity endpoint IC50 and the in vivo LD50 values for 347 chemicals as presented in the Registry of Cytotoxicity (Halle, 2003)).

Specialized Protocols

Test article concentrations may be adjusted as necessary to accommodate specific physical characteristics or client needs. Specialized protocols may be prepared as requested through consultation with the Study Director.


In 2007, ICCVAM recommended both in vitro test methods (3T3 NRU and NHEK NRU) as reduction alternatives to estimate the starting dose in the Up-and-Down Procedure and Fixed Dose Procedure for assessing acute oral systemic toxicity. Recommendations were accepted by U.S. Federal agencies, and OECD Guidance Document 129 for implementation of the test methods was published in 2010.