3T3 Neutral Red Phototoxicity(3T3 NRU PT)Testing Services

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What is Phototoxicity?

Phototoxicity - A toxic response from a substance applied to the skin which is either elicited or increased after subsequent exposure to light, or is induced by skin irradiation after system administration of a chemical.


The 3T3 Neutral Red Uptake Phototoxicity Assay (3T3 NRU PT) can be utilized to identify the phototoxic effect of a test substance induced by the combination of test substance and light and is based on the comparison of the cytotoxic effect of a test substance when tested after the exposure and in the absence of exposure to a non-cytotoxic dose of UVA/vis light. Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of the vital dye - Neutral Red.

Substances that are phototoxic in vivo after systemic application and distribution to the skin, as well as compounds that could act as phototoxicants after topical application to the skin can be identified by the test. The reliability and relevance of the 3T3 NRU PT have been evaluated and has been shown to be predictive when compared with acute phototoxicity effects in vivo in animals and humans.

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3T3 NRU PT Validation Studies


To evaluate the phototoxicity induced by the combined action of a chemical and light by using an in vitro cytotoxicity assay with the Balb/c 3T3 mouse fibroblast cell line.

Method Synopsis:

A slightly modified version of the protocol outlined in the OECD 432 guideline was followed for the validation of the 3T3 NRU Phototoxicity Assay. A Honle SOL 500 solar simulator was used with an H1 filter as the UV light source. Balb/c 3T3 cells were seeded in the central 60 wells of two 96-well plates and maintained in culture for 24 hours. The duplicate 96- well plates were then preincubated with eight different concentrations of the test article for 1 hour. Then one plate was exposed to a dose of 5 J/cm2 UVA while the other plate was kept in the dark. After UV irradiation, the treatment medium was replaced with culture medium and, after 24 hours, cell viability was determined by neutral red uptake for 3 hours.


The 3T3 NRU Phototoxicity Assay, an in vitro assessment of the phototoxic potential of a substance was validated following OECD 432 Guidelines. The validation of this assay involved testing eight chemicals with various phototoxic potential. The results of the definitive test were in accordance with the results obtained by the OECD, which were published in the 432 Guideline. Protoporphyrin IX, chlorpromazine (CPZ), anthracene, amiodarone and norfloxacin were classified correctly as phototoxins while hexachlorophene, sodium lauryl sulfate (SLS) and L-histidine were not phototoxic. These results demonstrate the validity of the adapted 3T3 NRU Phototoxicity assay used at MB Research.

Test Materials Not Soluble in Media?

EPARS - Enhanced Phototoxicity screening Assay in Reconstituted Skin

One of the key issues with the 3T3 NRU PT is that your test material must be soluble in cell culture media. The alternative is to utilize an animal model or to use the Enhanced Phototoxicity screening Assay in Reconstitued Skin(EPARS). EPARS is a novel assay using 3D Human Tissue Constructs/Equivalents developed at MB Research. EPARS allows for topical application of a test material (lotion, cream, etc.)

For more information about EPARS and our other Phototoxicity/Photobiology capabilities, please contact Client Services.

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