Background - 3T3 Neutral Red Uptake Phototoxicity Assay

General Background

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.

Basic Assay Outline

Seed 96-well plates with 3T3 cells

Dose 2 plates with 8 concentrations of Test Article. Incubate 37 °, 5% C02, 1 hr

Plate A: Expose to UVA 5 J/cm2.
Plate B: Keep in the dark, 50mins.

Return to 37 °, 5% C02, 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

Regulatory Background

The 3T3 NRU PT test 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.

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