The rainbow trout gill (RTgill-W1) cell line assay is a cytotoxicity test which has the potential to predict fish acute toxicity. Gill cells were chosen for this assay since gills are one of the main barriers between the fish and its surrounding water. Therefore fish gills are a major target site for environmental toxicants.
RTgill-W1 cells are seeded into 24-well plates and are incubated for 24 hours to attach and form a monolayer. Then they are exposed to a series of test chemical concentrations for a period of 24 hours. Exposure is performed with a minimal essential medium (L-15/ex) not containing bovine serum whereas normal L-15 cell culture medium containing bovine serum is used for the initial incubation. Cytotoxicity is determined using a combination of three endpoints representing cell viability: cellular metabolic activity measured with AlamarBlue, cell membrane integrity measured with 5-carboxyfluorescein diacetate acetoxymethyl ester...
The rainbow trout gill (RTgill-W1) cell line assay is a cytotoxicity test which has the potential to predict fish acute toxicity. Gill cells were chosen for this assay since gills are one of the main barriers between the fish and its surrounding water. Therefore fish gills are a major target site for environmental toxicants.
RTgill-W1 cells are seeded into 24-well plates and are incubated for 24 hours to attach and form a monolayer. Then they are exposed to a series of test chemical concentrations for a period of 24 hours. Exposure is performed with a minimal essential medium (L-15/ex) not containing bovine serum whereas normal L-15 cell culture medium containing bovine serum is used for the initial incubation. Cytotoxicity is determined using a combination of three endpoints representing cell viability: cellular metabolic activity measured with AlamarBlue, cell membrane integrity measured with 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and lysosomal membrane integrity measured with Neutral Red. Cell viability is expressed in % of the negative control. The respective EC50 value is calculated for each endpoint and EC50 calculation is based on measured exposure concentration. The most sensitive of the three endpoints is used for prediction of fish acute toxicity (based on 35 chemicals tested at the time of the submission EC50 would correspond to LC in fish).
Acute fish toxicity tests are e.g. part of the data requirements for REACH (10t or more) and are often required for safety testing of effluents. The RTgill-W1 cell line assay should support prediction of acute fish toxicity.
Chemicals with a specific neurotoxic mode of action and compounds that may be activated via metabolism were found to cause less toxicity in RTgill-W1 cells compared to fish.
