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

Objectivity in assessment of eye irritation 29 that the eye test system is subject to such wide variation that it will never be possible to make precise measurements of irritancy and that it will always be a subjective clinical appraisal, supported by measurements of corneal thickness and probably of intraocular pressure. Certain aspects of irritancy, such as the 'sting' phenomenon, which is probably of major commercial importance, cannot be measured in animal systems. REFERENCES 1 Draize, J. H., Woodard, G. and Calvery, H. O. Method for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Therap. 82 377-389 (1944). 2 Code of Federal Regulations. Tests for eye irritants. Title 16, Section 1500. 42 (1973). 3 Davies, R. E., Kynoch, S. R. and Liggett, M. P. Eye irritation tests - an assessment of the maximum delay time for remedial irrigation. J. Soc. Cosmet. Chern. 27 301-306 (1976). 4 Sebaugh, V. M., Osterberg, R. E., Hoheisel, C. A., Murphy, J. C. and Bierbower, G. W. A compara- tive study of rabbit ocular reaction to various exposure times to chemicals. Society of Toxicology JSth Annual Meeting Abs. 237 (1976). 5 Bayard, S. and Hehir, R. M. Evaluation of proposed changes in the modified Draize rabbit irritation test. Society of Toxicology 15th Annual Meeting Abs. 225 (1976). 6 Ballantyne, B., Gazzard, M. F., Swanston, D. W. and Williams, P. The ophthalmic toxicology of O-chlorobenzylidene malonitrate. Arch. Toxicol. 32 149-168 (1974). 7 Ballantyne, B. and Swanston, D. W. The scope and limitations of acute eye irritation tests. Current Approaches in Toxicology. Ed. B. Ballantyne 139-157 (1977). 8 Marzulli, F. N. and Ruggles, D. I. Rabbit eye irritation tests in collaborative studies. J. Ass. Off. Analyt. Chern. 905-914 (1973). 9 Kikkawa, Y. Normal corneal staining with fluorescein. Exp. Eye Res. 14 13-20 (1972). 10 Mishima, S. and Hedbys, B. J. Measurement of corneal thickness with a Haag-Streig pachometer. Arch. Ophthalrnol. 80 710-713 (1961). 11 Burton, A. B. G. A method for the objective assessment of eye irritatation. Fd. Cosmet. Toxicol. 10 209-217 (1972). 12 Conquet, P. H., Durand, G., Laillier, J. and Plazonnet, B. Evaluation of ocular irritation in the rabbit: objective versus subjective assessment. Toxicol. AppL Pharrnacol. 39 129-139 (1977). 13 Ballantyne, B., Gazzard, M. F. and Swanston, D. W. Effect of solvents and irritants on intra-ocular tension in the rabbit. J. Physiol. 226 128-148 (1972). 14 Heywood, R. and Walton, R. Intra-ocular pressure in eye irritancy studies. in press. 15 Laillier, J., Plazonnet, B. and le Douarec, J. C. Evaluation of ocular irritation in the rabbit: develop- ment of an objective method of studying eye irritation. Proc. European Society of Toxicology 17 336-350 (1975). 16 Wright, P. L., Ulsamer, A. G. and Osterberg, R. E. Effects of model eye irritants on corneal protein and water content. Society of Toxicology, 15th Annual Meeting Abs. 226 (1976). 17 Tonjura, A.M. Effects of benzylkonium chloride upon the corneal epithelium studied with scanning electron microscopy. Acta Ophthalmologica 53 358-366 (1975). 18 Ulsamer, A. G., Wright, P. L. and Osterberg, R. E. A comparison of the effects of model irritants on anaesthetized and nonanaesthetized rabbit eyes. Society of Toxicology, J6th Annual Meeting Abs. 143 (1977). 19 Heywood, R. Manifestations of ocular toxicity in laboratory animals. Ph.D. Thesis University of Bath (1977).