3T3 Neutral Red Phototoxicity Testing Services

Experience and Innovation.

MB Research has performed 100s of 3T3 NRU Phototoxicity tests and has experience in using this test with Pharmaceuticals, Cosmetics and Chemicals.

MB specializes in the conduct of Good Laboratory Practice (GLP) compliant phototoxicology assays. MB Research studies are designed to meet the requirements of the applicable regulatory agencies, or tailored to meet your specific needs.

MB Research offers more than 40 years experience in the conduct of acute, sub-chronic and chronic toxicology designed to meet regulatory guidelines.


The 3T3 Neutral Red Uptake Phototoxicity Assay (3T3 NRU PT) was developed and validated in a joint EU/COLIPA project from 1992-1997. In 1996, the 3T3 NRU PT was recommended by OECD as an in vitro approach for the assessment of chemicals phototoxicity potential. In April 2004, the 3T3 NRU PT protocol was finalized and adopted as the OECD 432 protocol, In Vitro 3T3 NRU phototoxicity test.

The 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.

Call for Quote or for more information about your testing needs:
US 1-215-536-4110 Or Contact MB.

Basic Assay Outline

  • Seed 96-well plates with 3T3 cells
  • Dose 2 plates with 8 concentrations of Test Article. Incubate 37 °, 5% CO2, 1 hr
  • Plate A: Expose to UVA 5 J/cm2.
  • Plate B: Keep in the dark, 50mins.
  • Return to 37 °, 5% CO2, 24 hrs
  • Add Neutral Red media, incubate 3 hrs. Rinse and Fix.
  • Read OD at 540 nm. Determine IC50UVA and IC50dark
  • Calculate Photo Irritation Factor Ratio; If > 6 then = phototoxin

Assay Specifics:

Test Model:


Test Materials Not Soluble in Media?

EPARS - Enhanced Phototoxicity screening Assay in Reconstituted Skin

EPARS is a novel assay using 3D Human Tissue Constructs/Equivalents. EPARS allows for topical application of a test material (lotion, cream, etc) that is not soluble in cell culture media. For more information about EPARS and our other Phototoxicity/Photobiology capabilities, please contact Client Services.

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