# New approach methodologies for metabolic disruption: a high-throughput human liver cell phospholipidosis assay

## TL;DR

This paper introduces a high-throughput screening method using human liver cells to detect chemicals that cause phospholipid accumulation, a condition linked to drug toxicity.

## Contribution

The study develops and validates a new in vitro assay using differentiated HepaRG cells for high-throughput screening of phospholipidosis inducers.

## Key findings

- The LipidTox assay in differentiated HepaRG cells reliably detects phospholipid accumulation in a 96-well format.
- The assay achieved 86% predictivity when compared to existing in vivo, in vitro, and in silico datasets.
- The method is suitable for identifying potential PLD inducers among diverse chemical groups.

## Abstract

Phospholipidosis (PLD) is the excessive accumulation of phospholipids within cells, which is, for example, observed in the liver following exposure to certain chemicals or drugs. Although the precise molecular mechanisms underlying PLD remain incompletely elucidated, it is documented that PLD is frequently induced by cationic amphiphilic compounds. To facilitate the identification of potential PLD inducers across diverse groups of chemicals, the development of novel high-throughput screening methodologies is imperative. Here, an in vitro human liver cell culture system was established for PLD screening, employing the fluorescence-based LipidTox assay in differentiated HepaRG cells in a 96-well format. Cells were treated with a selection of 35 compounds characterized as either PLD inducers or non-inducers, based on previously published in vivo data. Concentration-response curves of phospholipid accumulation were assessed and the results were compared with existing in vivo, in vitro and in silico datasets. The findings confirm the applicability of the LipidTOX assay in differentiated HepaRG cells as a reliable in vitro-based high-throughput screening method for PLD prediction, achieving an overall predictivity of 86%.

The online version contains supplementary material available at 10.1007/s00204-025-04254-2.