First-order toxicity assays can be used to rapidly screen test agents. Investigators in many laboratories have used cultured cell lines to obtain correlations between first-order assay end-points and in vivo eye irritation (Draize test) for a wide variety of compounds. Since validation is a key step in assay acceptance, it is important to understand which factors alter the responses of cell-line-based assays. In this study we examine: (1) the presence and configuration of a type I collagen gel; (2) the responses of epithelial (Sf-1-Ep) and fibroblast (Sirc and 3T3) cell lines; (3) the total glutathione content, ATP content, methionine incorporation, and neutral red absorption endpoint assays; (4) alcohol (C2-C8), surfactant (Tween 20), and heavy metal (NiCl) test agents; and (5) test agent exposure time (1 to 24 hr). The presence of a collagen gel and the cell type did not significantly affect endpoint assay R50 (test agent concentration that decreases assay response by 50%) values for a 1-hr exposure to hexanol. The ATP and glutathione endpoints (after 1-hr exposure) are able to distinguish between the relative in vivo toxicities of C2-C8 normal alcohols. All four endpoint assays detected sublethal damage, with the ATP and methionine endpoints being the most sensitive. The type of test agent affects the endpoint response, as shown by the lack of a glutathione R50 value for a 1-hr exposure to Tween 20 or NiCl. Even for a single test agent, endpoint assay R50 values may decrease continuously (ATP), decrease and then stabilize (glutathione), or remain unchanged (methionine incorporation) during a 24-hr exposure.