Phototoxicity test with 3T3 cells and reading of results with Neutral Red uptake. OECD 432: 2019 - In vitro 3T3 NRU phototoxicity test. 

Test Accredited by ENAC (Spanish National Accreditation Entity).

Test with the Certificate of Good Laboratory Practices (GLPs).

Phototoxicity is defined as a toxic response induced by a substance, which occurs or increases after body exposure to the sunlight, or which is induced by skin irradiation after systemic or topical cutaneous administration of a substance.

The in vitro 3T3 phototoxicity test with neutral red uptake is used to identify the phototoxic potential of a test substance caused by the excited chemical substance after exposure to light. The test evaluates the photocytotoxicity by the relative reduction in the viability of the cells exposed to the chemical in the presence of sunlight versus viability in the absence of light. The substances identified by this test are likely to be cytotoxic in vivo, after systemic administration and their distribution to the skin or after topical cutaneous application.

The 3T3 NRU in vitro phototoxicity test is based on the comparison of the cytotoxicity of a chemical substance when tested in the presence and the absence of exposure to non-cytotoxic doses of simulated sunlight. For this, the range of non-cytotoxic product concentrations must first be determined. The cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the neutral red vital dye when measured 24 hours after treatment with the chemical and been irradiated. Neutral red is a weak cationic dye that penetrates through cell membranes by a non-diffusion mechanism, accumulating intracellularly in lysosomes. The alterations of the lysosomal membrane surfaces that causes the fragility of the lysosomes and other irreversible changes, lead to a decrease in the uptake and fixation of the neutral red. Thus, it is possible to distinguish between viable, damaged or dead cells, which is the basis of this test.

The test uses Balb/c 3T3 cells that are kept in culture for 24 hours to obtain the cell monolayers. In each test, two 96-well plates are used with the cell monolayers, each preincubated with 8 different concentrations of the substance subjected to the test for 1 hour. The highest concentration of the chemical should be within physiological conditions, avoiding osmotic and pH stress. The guideline indicates that the maximum concentration of the product should not exceed 1000 µg/ml. For relatively insoluble substances that are not cytotoxic at the saturation point, the highest achievable concentration should be tested. Next, one of the two plates is exposed to the highest light irradiation that is not cytotoxic, while the other microplate is kept dark. In both plates, the treatment medium containing the substance under evaluation is replaced by culture medium and after 24 hours of incubation the cell viability is determined by the uptake of the neutral red dye. The cell viability is expressed as the percentage of cellular viability for each concentration with respect to the controls not treated with the evaluated chemical. To predict the phototoxic potential, the responses are compared to the concentrations of the substance obtained in the presence and absence of light irradiation, generally at the 50% inhibitory concentration (IC50), that is, the concentration that reduces 50% cellular viability, compared to untreated controls. With this value (IC50) the PIF (Phototoxic Irritation Factor) value will be calculated.

The test will be performed in two phases:

• Phase 1: Evaluation of the cytotoxicity of the product or extracted from the medical device.

• Phase 2: Phototoxicity study in products and concentrations, or extracted from medical devices, that have not demonstrated cytotoxicity in phase 1 at the concentration requested by the client.