Ocular instillation toxicity studies (OITSs) are one of general toxicity studies. Yet, OITSs have a unique characteristic that the test article is directly administered as eye drops to the target organ. Compared with general toxicity studies aiming systemic exposure, the study design of OITSs is somewhat distinctive in selecting test species, dosing formulation, administration volume/frequency and ocular examinations. After the administration of eye drops, the exposure level is high in the ocular surface, whereas the bioavailability in the eye balls, especially in the posterior segment, is low. In contrast to the general toxicity studies aiming systemic exposure, the absolute systemic exposure level in OITSs is generally low, while the systemic bioavailability is relatively high. These pharmacokinetic features determine the profiles of local and systemic toxicities in OITSs. Systemic toxicities are more often found in animals of relatively small body size, and are in most cases related with pharmacological actions. Current progress in ophthalmologic imaging technologies enables advanced safety evaluation using imaging biomarkers. Bioanalysis detecting drug levels present in blood in trace amount leads to a detailed safety assessment of systemic toxicity and yields accurate safety margins. Recognizing the peculiar characteristics of OITSs, toxicologists need to propose an appropriate study design and strategy of safety evaluation. Further discussion may be awaited on rationales for testing both sexes, and for conducting separated toxicity studies to evaluate systemic toxicity. This mini-review provides insight regarding current status and points to consider of OITSs.

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PLA can be a new surgical adjuvant to visualize the vitreous body during vitrectomy.

Several cationic-amphiphilic drugs such as chloroquine and amiodarone are known to induce phospholipidosis in the cornea by systemic administration. However, the characteristics of ophthalmological and pathological changes when phospholipidosis-inducing drugs are topically applied have not been well studied. This study was conducted to investigate the characteristics of corneal changes caused by topical application of chloroquine and amiodarone to Japanese white rabbits. The changes were evaluated by ophthalmological, histopathological, and ultrastructural examinations. An in vivo confocal microscopy was also applied to the chloroquine-treated corneas. In both chloroquine- and amiodarone-treated corneas, diffuse cloudiness was observed by slit-lamp biomicroscopy, and its transparency increased with duration of dosing. Confocal microscopy showed punctate dots in the corneal epithelium. Histopathologically, cytoplasmic vacuolation was found in the corneal epithelium and keratocytes in both chloroquine- and amiodarone-treated eyes. Furthermore, foamy cytoplasm of the corneal endothelium was observed in the chloroquine-treated eyes. Ultrastructural examination showed multi-lamellar inclusion bodies or membrane-like debris in the lysosome-like vacuoles in the cytoplasm of corneal cells, which is a characteristic of the lesions of phospholipidosis. These changes disappeared after a withdrawal period. Continuous dosing of chloroquine resulted in corneal erosion and focal corneal opacity as shown by gross observation and slit-lamp biomicroscopy. Confocal microscopy could detect the corneal changes prior to the appearance of these ophthalmological changes. The present study showed that phospholipidosis caused by ocular administration of chloroquine and amiodarone first induces reversible diffuse corneal cloudiness. Confocal microscopy is a useful method for monitoring induction of corneal phospholipidosis.

The anaphylatoxins C5a and C3a are involved in inflammation and host defense against bacteria, viruses, fungi and other pathogens. Anaphylatoxins play an important role in acute inflammatory reactions characterized by a series of vascular events, including an increased vascular permeability and recruitment of leukocytes. Although the function of C3a in disease is not as well defined as that of C5a, C3a has been shown to activate and be chemotactic for human eosinophils,2) mast cells 3) and rat neutrophils. 4) Humbles et al. 5) recently reported that, in addition to acquired immune responses, the innate system and complement (C3a in particular) are involved in the pathogenesis of diseases including asthma, a chronic inflammatory disease of the airways. Additionally, Fukuhara and Tsurufuji 6) and we 7) demonstrated that an extensive exudation occurred not only in the acute phase but also in the chronic phase of rat air pouch/carrageenan-induced inflammation. Therefore, we undertook the present studies to determine whether the complement anaphylatoxins C3a and C5a are present in the exudate of the chronic phase of rat air pouch/carrageenan-induced inflammation. The results reported here indicate that C3a and N-truncated C3a, but not C5a, are present in the exudate. MATERIALS AND METHODS Purification of C3a and N-Truncated C3aInflammation was induced by subcutaneous injection of 4 ml of a 2% (w/v) solution of carrageenan (Seakem 202; Marine Colloid Inc., U.S.A.) into a preformed air pouch on the back of male Wistar rats (body weight 170-200 g).6) On day 7 after the carrageenan injection, the pouch fluid was collected and centrifuged at 70000ϫg for 60 min at 4°C. The supernatant (day-7 exudate) was stored at Ϫ30°C until use for purification of C3a.All purification procedures except for reversed-phase HPLC (RP-HPLC) were carried out at 4°C. The frozen day-7 exudate (1000 ml) was thawed, its pH was adjusted to 4.5 with 9 N HCl, and the mixture was stirred for 2 h. After centrifugation at 13000ϫg for 60 min, the supernatant was brought to 38% saturation with (NH 4 ) 2 SO 4 and stirred for 3 h, and then centrifuged. Proteins in the supernatant were precipitated by addition of (NH 4 ) 2 SO 4 to 70% saturation. The resulting precipitate was separated by centrifugation at 13000ϫg for 60 min and dialyzed against 0.1 M sodium phosphate buffer (pH 6.0). The dialyzed solution was applied to a CM-cellulofine C-500 column (2.6ϫ47 cm; Seikagaku Co., Tokyo, Japan). The proteins bound to the column were stepwisely eluted with 0.1 M sodium phosphate buffer (pH 6.0) containing 0.15 M or 1 M NaCl. Proteins eluted with the buffer containing 0.15 M NaCl were dialyzed against 0.05 M Tris-HCl buffer (pH 7.0) containing 0.1 M NaCl, and the dialyzed solution was applied to a heparin-Sepharose CL-6B column (1.6ϫ26 cm; Amersham Pharmacia Biotech, Tokyo). Proteins were eluted between 0.6 M and 1.0 M NaCl during the course of a linear concentration gradient of NaCl from 0.1 M to 2.0 M. The basic proteins with a heparin-binding site, including C3...