Test No. 460: Fluorescein Leakage Test Method for Identifying Ocular Corrosives and
Severe Irritants
This Test Guideline describes an in vitro assay that may be used for identifying water
soluble ocular corrosives and severe irritants as defined by the UN Globally Harmonized
System of Classification and Labelling, Category 1. The assay is performed in a well
where a confluent monolayer of Madin-Darby Canine Kidney (MDCK) is used as a separation
between two chambers. It uses a fluorescein dye as marqueur. The test substance has
the potential to impair the junctions of the MDCK cells and thus to increase the monolayer¡¯s
permeability. Consequently the fluorescein passes through the monolayer and the fluorescein
leakage (FL) increases. The FL is calculated as a percentage of leakage relative to
both a blank control and a maximum leakage control. The concentration of test substance
that causes 20% FL (FL20, in mg/mL) is calculated and used in the prediction model
for identification of ocular corrosive and severe irritants. The cut-off value of
FL20 to identify water soluble chemicals as ocular corrosives/severe irritants is
¡Ü 100mg/mL. The FL test method should be part of a tiered testing strategy.
Published on October 09, 2017Also available in: French
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