Validation of the use of zebrafish larvae in visual safety assessment

The use of zebrafish (Danio rerio) larvae was investigated to predict adverse visual effects and to establish the potential application of this organism in early drug safety assessment.

Following a comparison of the effects of 4 compounds in TL and WIK strains of zebrafish larvae, a blinded validation set of 27 compounds was tested on WIK strain of larval zebrafish in the optomotor response (OMR) assay. Selected compounds were also tested in the optokinetic response (OKR) and locomotor assays. Larvae were exposed from 3-8 days post-fertilisation (d.p.f.) by immersion in embryo culture media (E3) containing the compound in 1% DMSO (v/v). At 8 d.p.f. toxicity was assessed and the OMR or OKR assays were undertaken at non-toxic treatment levels. Compounds were then rated as 'red', 'amber' or 'green' according to their effects on visual function prior to unblinding of the identities of the test compounds.

Overall, the OMR assay revealed a good concordance between the effects of compounds in WIK strain zebrafish with the data available from other in vivo and in vitro models or the clinic: thirteen out of nineteen positive compounds produced the expected effect while six of the eight negative compounds were correctly predicted. This gave an overall predictivity of 70% with a sensitivity of 68% and a specificity of 75%. The two false positive compounds were further tested in locomotor and optokinetic response assays and it was shown that a motility defect, rather than an effect on vision, had given rise to the positive result in the OMR assays. Therefore, the OMR assay would best be employed with other techniques to identify false positives. Further studies on two of the false negatives at higher concentrations suggested that the initial concentrations tested were too low. Therefore, it should be ensured that the maximum tolerated concentration is tested in the OMR assay. A comparison of four standard compounds in the OMR assay in WIK and TL zebrafish wild type strains revealed no difference in sensitivity between the strains.

Overall, these results suggest that the OMR assay in zebrafish could be useful in predicting the adverse effects of drugs on visual function in man and would support its potential as a screen for 'frontloading' safety pharmacology assessment of this endpoint in vivo.