Objectivity in assessment of eye irritation 27 be credited with first realising the usefulness of this parameter in assessing irritancy. Initially, it appeared to be a sophisticated and objective measure of irritancy, free from observer bias, but unfortunately, with experience the limitations become apparent. It is important that the measurement of thickness is made at the apex of the cornea. It is unfortunate that the cornea is not uniformly thick, nor does it uniformly respond to irritation. If sloughing of the epithelium occurs the measurement becomes meaningless. There is again an observer bias as to the point on the cornea which one chooses to measure. Nevertheless, it has been shown with commercial shampoos, Burton (11), and a variety of industrial solvents, Conquet et al. (12), that a highly significant correlation can be shown between the total Draize score and the corneal thickness measurement. It must be concluded that this measurement of corneal thickness can only supplement the clinical observations. INTRA-OCULAR PRESSURE Ballantyne, Gazzard and Swanston (13) showed that a rise in intra-ocular tension is one of the earliest indications that a compound is producing eye irritation. Furthermore, they suggested that there might be a relationship between the rise in tensiola and the severity of the subsequent reaction. There are two indirect methods of measuring intra-ocular pressure. Firstly, there is the indentation tonometer, by which a weighted plunger is applied to the cornea, the most common piece of apparatus used being the Schiotz tonometer. The second and most applicable technique is that of applanation tonometry, which measures the force required to produce a degree of flattening. The use of the Schiotz tonometer is often impractical, because swelling of the cornea cushions the tonometer footplate and a satisfactory measurement cannot be made. To date, there is little published data to support the use of applanation tonometry for measuring ocular irritancy but current investigations - Heywood and Walton (14) - will indicate the potential of this technique. CORNEAL CURVATURE Measurement of corneal curvature is precluded by the effects of irritants on the pre- corneal film, because any disturbance of this film and the superficial epithelial cells of the cornea prevents lining up of the mires of the ophthalmometer and making accurate measurements. CONJUNCTIVA• AND CORNEAL WEIGHT Conjunctival and corneal weights were found to be precise measurements of irritation by Laillier, Plazonnet and Douarec (15), who investigated many organic solvents. For control animals, the percentage dry weight was 26-4•o-½0'8•o, but even with the most severe irritants, this never fell below 10•o. The water content of the cornea does not show a direct relationship to opacity, Wright, Ulsamer and Osterberg (16). EFFECTS ON CAPILLARY PERMEABILITY Changes in capillary permeability in the conjunctiva and the blood/aqueous level can be demonstrated using the Evans blue dye technique. Laillier et al. (15) have shown change in capillary permeability to be one of the first events in eye irritation.

28 R. Heywood and R. W. James HISTOLOGICAL INVESTIGATION At the end of each study, the eye and its adnexa should be taken for histological examina- tion. The techniques used should be such as to reduce fixation artefact to a minimum. The normal cornea should show microvillae in the superficial cells of the corneal epithelium, microtubules in Descemet's membrane and vesicles in the endothelium, indicating active transport systems. There should be no oedema and the collagen fibres in the stroma should be of normal size and show no distortion. In the conjunctiva, there should be normal microvillae and the epithelium cells should be rich in endoplasmic reticulum. Normal goblet cells are of considerable importance, as they are responsible for the mucin in the precorneal film. Alterations in any of these parameters must be considered of significance. Tonjum (17) investigated the corneal epithelium of rabbits and vervet monkeys after treatment with benzylkonium chloride and demonstrated that the superficial cells were showing small holes and losing microvillae within 2 min of application of the irritant. THE USE OF ANAESTHETICS The present tests make no provision for the use of anaesthetics. Ulsamer, Wright and Osterberg (18), working with acetic acid, phenol, sulphuric acid, ammonia and sodium hydroxide, together with the anaesthetic butacaine sulphate (2%) measured the opacity, redness response, corneal water content, dry weight and electrophoretic protein patterns in groups of New Zealand White rabbits. Neither the opacity scores nor the moisture content produced clear-cut patterns of statistical differences, although with 10•o acetic acid and 10• ammonia, the unanaesthetised eyes had lower scores. The non- anaesthetised cornea had a lower water content following the administration of 5% acetic acid and 1% ammonia and greater water content following the administration of 3% sodium hydroxide solutions. Using 10% sodium lauryl sulphate as the irritant and 0.5% proparacaine as the anaesthetic in the New Zealand White rabbit, Heywood (19) showed that no statistically significant differences could be detected between the anaesthetised and unanaesthetised cornea. Although there was some evidence that the intensity of reaction was increased following anaesthesia, there was no evidence of prolonged effect. In the monkey, however, applying irritants to the anaesthetised corneal sac had a very marked effect in which all reactions were intensified and prolonged. The results indicate that, in the rabbit, the use of a local anaesthetic should be considered when carrying out eye irritation tests, but in the monkey the use of anaes- thesia is contraindicated on both scientific and humanitarian grounds. DISCUSSION On reviewing the literature, there appears to be no data which establishes convincingly a correlation between the Draize score and the objective irritancy measuring tests, although such a correlation has been established with certain shampoos and industrial solvents. However, Heywood (19), working with a variety of methods, treatment con- centrations and regimes, showed that when correlations could be established these were between group mean values and could not be shown to occur consistently in individual animals. To draw any conclusion from the available data is difficult, for it would appear