Comparison of in vitro transcriptomic points of departure with fish acute and chronic toxicity values: A case study of rainbow trout cells exposed to pesticides.

There is growing interest in transcriptomic points of departure (tPOD) values from in vitro experiments as an alternative to animal test method. The study objective was to calculate tPODs in rainbow trout gill cells (RTgill-W1 following OECD 249) exposed to pesticides, and to evaluate how these values compare to fish acute and chronic toxicity data. Cells were exposed to one fungicide (chlorothalonil), ten herbicides (atrazine, glyphosate, imazethapyr, metolachlor, diquat, s-metolachlor, AMPA, dicamba, dimethenamid-P, metribuzin), eight insecticides (chlorpyrifos, diazinon, permethrin, carbaryl, clothianidin, imidacloprid, thiamethoxam, chlorantraniliprole), and OECD 249 positive control 3,4-dichloroaniline. Pesticide concentrations in wells were modeled with IV-MBM EQP v2.1. Sequencing libraries were prepared with UPXome, and tPODs calculated with ExpressAnalyst. The method identified 14,449 unique genes, with 1,115 genes having >5 counts in the 576 samples sequenced. For all chemicals, tPODs were derived and tPOD mode values ranged from 0.0004 to 125µM with an average of 36µM. There were significant correlations between tPOD mode values (x-value) and EC50s from RTgill-W1 cells (y = 0.92x+1.2, R2=0.9, p < 0.00001; n = 11), rainbow trout acute toxicity LC50s (y = 0.81x+0.8, R2=0.63, p < 0.0001; n = 20), fish chronic sub-lethal effect concentrations (y = 0.53x-0.2, R2=0.4, p = 0.009; n = 16) and fish chronic lethal effect concentrations (y = 0.64x-0.023, R2=0.59, p = 0.0013; n = 14). Bland-Altman plot analyses of these comparisons also showed good agreement. Pathway-level benchmark doses were calculated when statistical requirements were met, and only possible for four pesticides. These findings support the notion that tPODs from short-term in vitro studies may be comparable to effect concentration data from in vivo studies of fish exposed for chronic durations.