Chemicals, cosmetics, and pharmaceuticals have to be assessed for their irritancy potential and risk to the human eye. However, the only method accepted worldwide by regulatory authorities for the assessment of acute eye irritation potential is the Draize rabbit test,
1 which has been criticized by animal welfare advocates and its relevance, validity, and precision challenged because of the variability and low predictiveness of the human response.
2 3 This test is mainly based on scoring of observed macroscopic changes in the rabbit cornea, conjunctiva, and iris. Various scoring systems are currently accepted (Maximum Average Score [MAS], Modified Maximum Average Score [MMAS], and the Globally Harmonized System). Among the in vitro alternatives accepted in 2007, the bovine corneal opacity and permeability (BCOP),
4 5 6 7 and the isolated chicken eye (ICE)
8 9 assays were found to adequately predict severe irritancy but were not recommended to identify mild to low irritants. Therefore, the principal challenge remains the development of alternative methods suited for assessing nonsevere irritants and nonirritants. The reconstructed three-dimensional (3D) model of human corneal cells (HCEs), supplied by SkinEthic Laboratories (Nice, France), was found to resemble the corneal epithelium of the human eye in morphology and thickness.
10 It was proposed as a useful alternative model to the classic Draize test
11 12 for the assessment of eye irritation potential of chemicals and cosmetic products. The toxicokinetic approach described by Doucet et al.
12 13 was based on the MTT test procedure previously published on 3D-reconstituted epidermal and corneal models. The MTT test has already been shown to constitute a rapid and cost-effective method of screening for surface toxicity of topical agents using monolayer cell cultures
14 and the procedure used on 3D-models provided a good correlation between in vitro results and MMAS values that correspond to the MAS obtained after 24 hours or more in vivo. However, because the MTT solution was placed under the construct, it could penetrate only two or three layers from the basal side, yielding results that may not correlate with histomorphologic findings in the superficial layers. This procedure may therefore underestimate any mild toxic effect occurring at the apical surface of the constructs. In the literature, systematic histologic analysis and multiple endpoint analysis (MEA) were proposed as practical responses to prevent the occurrence of false-negative MTT results.
15 16 17 Although MEA is a valuable approach to determine the mechanisms of eye irritation, the MTT procedure should be optimized separately so as not to neglect the cellular events occurring in the superficial layers. We believe that a small change in the MTT procedure could lead to increased sensitivity, not only making this procedure better suited for risk prediction in case of accidental exposure to the eye, but also adapting it to the prediction of low to very low irritant potential, especially when products are used repeatedly over long periods. The implementation of these very sensitive tools to predict eye irritation is critical for ophthalmologists treating patients who may be exposed to long-term induced toxicity of substances used at low concentrations in ophthalmic preparations. One of them is the widely used eyedrop preservative benzalkonium chloride (BAK), whose toxic and inflammatory effects have been demonstrated in clinical
18 19 20 21 22 23 24 in vivo,
20 21 25 26 and in vitro studies.
27 28 29 BAK is known to induce oxidative effects
30 and caspase-dependent and -independent apoptosis counteracted by an autophagic process.
31 Chronic use of preservative is responsible for apoptosis of conjunctival cells and conjunctival inflammation, which has demonstrated negative effects (e.g., on the efficacy of glaucoma surgery).
19 32