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Phototoxicity: 3T3 Neutral Red Uptake (NRU) Phototoxicity test

This assay is designed to assess cytotoxicity of test substances in the presence or absence of UV light. In this case, cytotoxicity is used as a proxy for acute phototoxicity, a chemically-induced skin irritation, requiring light for initiation of the effects.

The method is a cell based assay that uses 3T3 cells, with cytotoxicity assessed via neutral red uptake following exposure to the test substance (and light or not).

The test method has been approved for regulatory purposes to predict acute phototoxicity effects in animals and humans in vivo. It is the subject an OECD test guideline (No 432) and has been included in a number of EC regulations relating to the evaluation of chemical safety.

A number of validation studies have been carried out on the method and these have been reviewed by the EURL ECVAM Scientific Advisory Committee (ESAC) and published in peer-reviewed journals.

The results and full reports will be soon available on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.


[collapsed]Phototoxicity (photoirritation) is defined as a toxic response that is elicited after the initial exposure of skin to certain chemicals and subsequent exposure to light, or that is induced by skin irradiation after systemic administration (oral, intravenous) of a chemical substance.

If a chemical absorbs UV or visible light, there is a chance that the absorbed energy can cause molecular changes that result in the chemical becoming toxic.

Determining phototoxicity potential becomes important when a chemical is intended for human use.[/collapse]

The 3T3 Neutral Red Uptake (NRU) Phototoxicity test

[collapsed]The 3T3 NNeutral Red Uptake (NRU) Phototoxicity test is based on an immortalised mouse fibroblast cell line called Balb/c 3T3.

The method is based on a comparison of the cytotoxicity of a chemical when tested in the presence or absence of exposure to a non-cytotoxic dose of simulated solar light (i.e. ultra-violet light).

Cytotoxicity in this test is expressed as a concentration-dependent reduction of the uptake of the vital dye neutral red when measured 24 hours after treatment with the test chemical and irradiation.

The test chemical together with the irradiation may alter the cell surface and in effect may result in a decreased uptake and binding of the neutral red dye.

Differences in this uptake can be measured with a spectrophotometer, which allows the distinction and quantification between viable, damaged or dead cells.[/collapse]

Animal testing replacement

[collapsed]The method has been validated (see below) for the assessment of whether chemicals have the potential to cause acute photo-toxic effects in animals and humans in vivo.

The test is not designed to predict other adverse effects that might occur through the combined action of a chemical and light. This might include photogenotoxicity, photoallergy or photocarcinogenicity.

The method cannot assess potency and is not designed to address indirect effects of phototoxicity, the effects of metabolites or the effects of mixtures.

In comparison to a direct animal-based test, the method is comparatively narrow in what it can test for (cellular cytotoxicity through the action of a chemical and light combined) and is not designed to fully replace animal-based testing.

Nevertheless, in combination with other methods and within appropriate testing strategies, it may reduce the number of animals needed to assess phototoxicity.

Specifically, negative outcomes with this test strongly suggest negative outcomes would be achieved in vivo and likewise the same would be the case for positive outcomes.

Testing on animals such as guinea pigs, rabbits or rodents were accepted methods but these have now been replaced by a range of validated in vitro assays.[/collapse]

Validation studies and outcomes

[collapsed]The 3T3 Neutral Red Uptake (NRU) assay for phototoxicity has been subjected to a series of validation studies.

Between 1992 and 1997, the assay was refined and evaluated as part of the EU/COLIPA international validation study on in vitro tests for phototoxic potential.

Based on the success of this validation study, ESAC unanimously endorsed a statement in 1998 that the method was scientifically validated and ready to be considered for regulatory acceptance.

A second validation study was also carried out in 1997 to evaluate the method specifically in terms of selected UV filter chemicals from Annex VII of EU Directive 76/768/EEC. ESAC subsequently endorsed the validity of the test with respect to these chemicals.

The method has subsequently received regulatory acceptance and is included in a number of directives (see DB ALM protocol No 78 for more details).

Furthermore, the method has been adopted as OECD test guideline No 432.[/collapse]