In Vitro 3T3 NRU Phototoxicity Test

Phototoxicity is a toxic response from a substance applied to the body which is either elicited or increased after subsequent exposure to light, or that is induced by skin irradiation after systemic administration of a substance. The phototoxicity test is used to identify the phototoxic potential of a test substance induced by the excited chemical after exposure to light. The assay can be performed in accordance with Good Laboratory Practices. The experiment is designed to follow the current OECD 432, “in vitro 3T3 NRU phototoxicity test” (2004) Guideline.

Typically, BALB/c 3T3 cells (clone 31) are seeded into two 96-well plates and maintained in culture for 24 h (~ 1 doubling period) to form a semi-confluent monolayer. Two 96-well plates per test chemical are pre-incubated with eight different concentrations of the test substance for 1 h. Afterward one of the two plates is kept in the dark whereas the other is exposed to the highest non-cytotoxic irradiation dose. The cells are irradiated with a mix of visible light, UVA and UVB light that simulates the solar spectrum. The agent chlorpromazine is used as a positive control. In both plates the treatment medium is then replaced by culture medium and after another 24 h of incubation cell viability is determined by Neutral Red uptake. Cell viability is expressed as percentage of untreated solvent controls and is calculated for each test concentration. To predict the phototoxic potential, the concentration responses obtained in the presence and in the absence of irradiation are compared, usually at the IC50 level (i.e. the concentration producing 50 % reduction of viability). The test evaluates photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light. Substances identified as positive by this test are likely to be phototoxic in vivo following systemic application and distribution to the skin, or after topical application.

Experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review within 4-6 weeks after the experiment start date. Documents will be archived for six years.

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