First-order toxicity assays for eye irritation using cell lines: parameters that affect in vitro evaluation

Area of Science:

• Toxicology
• In vitro assays
• Cell culture

Background:

• First-order toxicity assays offer rapid screening of test agents.
• Cell-line-based assays are used to correlate with in vivo eye irritation (Draize test).
• Assay validation requires understanding factors that alter cell-line responses.

Purpose of the Study:

• To examine the impact of collagen gel, cell type, and assay endpoints on toxicity assay results.
• To evaluate the sensitivity of glutathione, ATP, methionine incorporation, and neutral red absorption assays.
• To assess the influence of different test agents (alcohols, surfactant, heavy metal) and exposure times on assay outcomes.

Main Methods:

• Cultured epithelial (Sf-1-Ep) and fibroblast (Sirc, 3T3) cell lines were used.
• Assays included glutathione content, ATP content, methionine incorporation, and neutral red absorption.
• Test agents comprised alcohols (C2-C8), Tween 20, and NiCl, with exposure times from 1 to 24 hours.

Main Results:

• Collagen gel presence and cell type did not significantly alter R50 values for hexanol exposure.
• ATP and glutathione endpoints distinguished in vivo toxicities of C2-C8 alcohols after 1-hour exposure.
• ATP and methionine endpoints were most sensitive in detecting sublethal damage across various agents and times.

Conclusions:

• Cell-type and collagen gel had minimal impact on hexanol toxicity R50 values.
• ATP and glutathione assays show promise for predicting alcohol toxicity, supporting validation of these methods.
• Endpoint assay responses vary significantly based on test agent type and exposure duration, necessitating careful selection and interpretation.