Histopathology in the isolated chicken eye test and comparison of different stainings of the cornea

► PAS appeared to be the superior staining method for the chicken cornea. ► Trichome, AZAN and EVG were less successful in staining the critical parts of the (chicken) cornea. ► Bowman’s membrane is present in the chicken and in the human cornea, but not in the rabbit cornea. ► Histopathology of the treated cornea in the ICE test is confirmative and useful for evaluation of borderline effects. ► Besides Depth-of-Injury, immunostaining of (chicken) corneal limbal stem cells might be useful to determine reversibility. The isolated chicken eye (ICE) test, developed at our Institute, is accepted by the OECD for identification of severe eye irritants. The OECD ICE Guideline (No. 438) encourages preservation of the treated eyes for possible histopathology of the cornea, which is believed to strengthen evidence of absence or presence of irritation and to help clarify borderline effects by assessment of the corneal Depth-of-Injury. Histopathology of the cornea in addition to the normal slit-lamp microscope assessment of corneal effects has already been performed routinely in ICE tests at our Institute, using two standard stainings (H&E and PAS). In this study, three other stainings (AZAN, EVG and Trichrome), more specific for collagen-rich membranes such as basement- and Bowman’s membranes were examined with corneas exposed to four model compounds ranging from non- to severely irritating (corrosive). PAS appeared to be the superior staining method. Surprisingly, the well-known eye corrosive sodium hydroxide (NaOH, solid) did not visibly compromise the integrity of Bowman’s or the basement membrane. Based on our experience, histopathology of the treated cornea is confirmative in relation to the standard assessment of eye irritation by slit-lamp observation in the ICE and in certain cases can help to evaluate borderline effects. Besides establishing the depth of injury, additional investigation of corneal limbal stem cell damage after chemical exposure might be appropriate to determine reversibility or irreversibility of eye effects.