IIVS | 3T3 Neutral Red Uptake (OECD 432)
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3T3 Neutral Red Uptake (OECD 432)

The 3T3 Neutral Red Uptake (NRU) Phototoxicity assay is a 96-well cytotoxicity-based assay that utilizes normal BALB/c 3T3 mouse fibroblasts to measure the concentration-dependent reduction in neutral red uptake by the cells after exposure to a test material either in the presence or absence of UVA light. Duplicate 96-well monolayers of 3T3 fibroblasts are exposed to serial dilutions of a test material. One of the plates is exposed to 5 J/cm2 UVA while the other plate is kept in the dark. To assess viability, the neutral red uptake (NRU) by cells exposed to the test chemical in the presence of UVA exposure is compared to the NRU by cells exposed to the test chemical in the absence of UVA exposure.

Quick Facts

Assay Model: 3T3 cells seeded in duplicate 96-well plates

Endpoints: calculated using Phototox 2.0 software (supplied by ZEBET)

  1. IC50 (the concentration of test material that causes a 50% decrease in viability, relative to the solvent control)
  2. PIF (Photo-Irritation-Factor) compares the IC50 of treated cells in the presence of UVA exposure to the IC50 of treated cells in the absence of UVA exposure.
  3. MPE (Mean Photo Effect) compares the response curve of treated cells in the presence of UVA exposure to the response curve of treated cells in the absence of UVA exposure.

Specialized protocols may be prepared as requested through consultation with an IIVS Study Director.

3T3 NRU Photoxicity Assay: Phototoxicity assay - overview of the procedure. Duplicate plates of test chemical 3T3 cells are exposed in the presence or absence of UVA.
Example of chlorpromazine (positive control) dose responses in presenceand absence of UVA.
Applications

The 3T3 NRU phototoxicity assay is generally used to test the phototoxic potential of chemicals and ingredients, rather than final products and formulations.

Test Material Compatibility

The assay is 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, an initial solubility test is performed to find the most suitable solvent when the solubility of a test chemical is unknown.

Test materials that are found to be insoluble or otherwise incompatible with this test system (3T3 NRU Phototox) may be tested using our 3-D Reconstructed Human Epidermis Phototoxicity assay.

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.

Alternate assays measuring the potential for phototoxic action may be employed using 3-dimensional reconstructed human epidermal tissue models. Please contact us for more information.

Validation Status

OECD TG432

Phototoxicity Comparisons: Comparison of the responses between phototoxic and non-phototoxic test materials.

Phototoxicity Comparisons: Comparison of the responses between phototoxic and non-phototoxic test materials.

Step-by-Step

  1. Seed
  2. Dose & UVA
  3. Test Article Removal & Rinsing
  4. Addition of Vital Dye
  5. Addition of Solvent
  6. 96-well Plate Reading
Step 1: Seed

The cells to be used for the 96-well assays are stored in a liquid nitrogen freezer. When ready for use, the cells are thawed, resuspended, and transferred to a culture flask, where they will grow and multiply until optimal conditions have been achieved.

Trypsin solution is added to the flask to remove the cells. The cells are resuspended in medium, counted, and “seeded” into 96-well plates using a multichannel pipet.

Cell storage
Cell amoule
Cell culture
96 well plate cultures
Step 2: Dose & UVA

The test material is serially diluted to make a range of doses.

The test article dilutions are added to the inner wells containing cells.

Each plate also contains 12 negative, or solvent, control wells which are used to determine % of control viability.

One plate dosed with test material is then exposed to UVA light while the other remains in the dark at room temperature.

Serial dilution series
Dosed 96 well plate
Solar simulator
Solar simulator
Step 3: Test Article Removal & Rinsing

The test material is removed by decanting. The plates are rinsed with a buffered saline solution to remove any residual test material.

Test material removal
Test material rinsing
Rinsate removal
Step 4: Addition of Vital Dye

A solution of Neutral Red, a vital dye, is added to the 96-well plate. The plates are incubated at standard culture conditions to allow neutral red uptake by the cells.

Neutral red addition
Neutral red uptake
Step 5: Addition of Solvent
Solvent addition

Solvent addition

After the 3 hour neutral red incubation, excess neutral red is decanted and solvent is added to all wells. The solvent extracts the neutral red dye contained within the cells.

Step 6: 96-well Plate Reading
Optical Density Determination

Optical Density Determination

The 96-well plates are placed on a plate shaker to fully extract the neutral red and evenly distribute the dye in each well.

A spectrometer measures the absorbance of each sample at a specific wavelength.

The absorbance values (optical density) are then used to determine the viability of each well by comparing the optical density of the each test material treated well compared the negative (or solvent ) control wells.

#3 shows phototoxicity (no cytotoxicity) #4 shows phototoxicity and cytotoxicity

Assays of Interest