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Ophthalmologic examination in systemic toxicity studies: an overview B. Kuiper1, M. H. Boeve2, T. Jansen3, M. E. Roelofs-van Emden4, J.W. G. M. Thuring4 & M. V. W. Wijnands5 1Notox Safety and Environmental Research BV,'s-Hertogenbosch,The Netherlands, 2Utrecht University, Faculty of Veterinary Medicine, Department of Clinical Sciences of Companion Animals, Utrecht, The Netherlands, 3Janssen Research Foundation, Beerse, Belgium, 4NVOrganon, Oss,The Netherlands and sTNO Nutrition and Food Research, Zeist,The Netherlands Summary The procedures of ophthalmologic examination used by the authors during systemic toxicity studies in laboratory animals are presented in this paper. The present international guidelines with respect to the requirements for ophthalmological examination are given in an overview. A list of proposed keywords is included which may be used in describing ocular changes. Keywords Ophthalmoscopej mydriatic agentj systemic toxicity studiesj restrainingj fundu- scopy; terminology; guidelines; morphology; physiology Ophthalmologic examination has become a routine procedure in systemic toxicity studies, since it is required by many authorities. Most veterinary surgeons are trained in ophthalmologic examination of companion animals but they have little experience with this type of examination in laboratory animals. Veterinary surgeons in a number of industries in The Netherlands and Belgium, involved in toxicity testing, felt the need to pool their knowledge and experience in order to establish a sound handling of all aspects of ophthalmologic examination. This concerned equipment, procedures, terminology and the various guidelines for systemic toxicity studies. The results of these discussions are pre- sented in the present paper. Materials and methods Equipment Schirmer tear test strips Mydriatic agent Correspondence to: Dr B. Kuiper. Catsstr. 9, 7009 JK Doetinchem, The Netherlands Accepted 10 July 1996 Handheld slitlamp biomicroscope (KOWA SL-51 Tonometer (Tono-Pen, Digilab 30R or Mackay-Marg) Ophthalmoscope (direct or indirect type) Handheld funduscamera (e.g.KOWARC-21 Restraining All methods of ophthalmological examina- tion routinely used in systemic toxicity studies with dog, cat, minipig, mouse, rat and rabbit may be applied in the conscious animal. Non-human primates are usually examined while sedated or under general anaesthesia. Dogs and cats may be examined while held by a technician. Minimal restraint is necessary to keep the animal in a standing, sitting or sphinx position. A well designed sling (Panepinto et 01. 19831 is very con- venient when examining minipigs. Re- straint of mice (Fig. 1) may be difficult, because of the small size of the animals and their tendency to bite. Manual restraint is commonly used. However, care should be taken to minimize the risk of suffocation. Gentle restraint of rats (Fig. 2), preferably LaboratoryAnimals (1997) 31, 177-183 178 Fig1 Direct ophthalmoscopy in the mouse Fig2 Slitlamp-biomicroscopy in the rat Fig 3 Indirect ophthalmoscopy in the rabbit Kuiper et al. on a table with adjustable height, by an experienced technician, is sufficient to per- form the routine examinations described below, provided that the animals are accustomed to manipulation. A restraining device for ophthalmologic examination has been described (Anderson et al. 1991), but the necessity of the device is doubted by us and other authors [Lee 1992). Rabbits (Fig.3) are positioned in such a way that the examiner's eyes are below the animal's eye during funduscopy,in order to facilitate visualization of the optic disc region. A grid may be placed on the table for the animal's convenience. Procedures External inspection The eye and the peribulbar structures are observed macro scopically. Tear production test All manipulation with or medication of the eye will influence tear production. For this reason the Schirmer tear test (STT) should be performed before any other manipulation. STT strips are used to assess tear production in man and animals. Reference values in a number of species are listed in Table 1. Reference values in animals are based on performance of STT without topical anaesthesia (Rubin et al. 1965, Gelatt et al. 19751.Narrow strips may be used in the rat (Weisse et al. 1978),but we do not include the STT in this species. Tear production in animals is measured [generally unilaterallyl during one minute. Pupillary reflexes The evaluation of pupil- lary reflexes is performed in a dimly-lit room. Table 1 Reference values Schirmer tear test Mean SD Species Strain (mm) (mm) Reference Monkey Macaca 15 3.7 Jaax et al. 1984 mu/atta Rabbit NZW' 4.97 2.4 Abrams et a/. 1990 Cat Domestic 16.9 5.7 Veith et al. 1970 short hair Dog Various 19.8 5.3 Rubin et a/. 1965 'New Zealand White Ophthalmologic examination in systemic toxicity studies The first action in this test consists of the estimation of the pupil width. In rat toxicity studies the pupillary reflexes are not rou- tinely assessed. Direct and consensual pu- pillary reflexes are evaluated in dog, cat and rabbit studies. Pupillary dilation Agents lmydriatics) which may be used for pupillary dilation by topical application are: atropine sulphate (0.5-1%), homatropine (2%) and tropicamide (0.5%) (Mydriaticum, Bournonville-Pharma). Full mydriasis occurs in most species within 15-30 min. The latter two mydriatics are short-acting and loose their effect within a few hours. The effect of atropine sulphate is long- acting. It exerts a prolonged duration of activity (>24h) which may lead to a deleterious effect on the retinal function. Its use is therefore not recommended. Overexposure to light may result in retinal degeneration in sensitive strains (Williams et a1. 1985, Schlingmann et a1. 1993). Moreover, in some species, atropine sul- phate may act as a mucosal irritant and may result in profuse salivation. After instillation of atropine sulphate, mydriasis may fail to occur in rabbits, due to the presence of atropinesterase activity (Van Zutphen 1974, Lee 1983). Slitlamp biomicroscopy The anatomy and the localization of abnormalities in the anterior eye segment are investigated (Seve 1985).Commonly, in biomicroscopy a hand- Table 2 Reference values intraocular pressure Mean SO [mmHg] [mmHg] Reference Man 15 2.5 Gelatt 1991 Non-human 15 Gelatt 1991 primate Rabbit 24.4 1.3 Poyer et al. 1992 Rabbit (NZR)' 19.3 1.3 Knepper et al. 1985 Dog 21.4 2.1 Gelatt 1991 Dog 15.5 1.1 Wilkie et al. 1991a Cat 17.1 1.1 Wilkie eta/. 1991b 'New Zealand Red 179 held slitlamp biomicroscope is used IKOWAI. Experience with this method has been obtained in many species. The magnification should be at least 15 times. This examination is performed in a darkened room. Tonometry Measurement of the intra- ocular pressure (lOP) or tonometry has been performed in many animal species, includ- ing the rat (Gelatt 1991). However, tono- metry is not routinely performed in systemic toxicity studies. A local anaes- thetic (Novesin [Chibret]) is applied before measurement of the lOP. Calibration of the equipment based on values in one species does not guarantee that the values obtained in a different species are valid, because the specificity of the method mainly depends on the curvature and the structure of the cornea and sclera. Some reliable applanation tonometers are available (e.g. Tono-Pen, Mackay-Marg or Digilab 30R). lOP in healthy rats ranges between 15 and 30mmHg with the Tono-Pen (Moore et a1. 1993). Funduscopy For funduscopy, direct and indirect ophthalmoscopes are suitable, although the indirect system is preferred for its wider view and better convenience to the examiner. A large assortment of reliable ophthalmoscopes are marketed nowadays. This examination is performed in a darkened room. Photography Photography is of great importance in the documentation of both normal and pathological morphology (Bo- kobza 1988, Williams 1992, Barnett 1990). KOWA RC-2 or KOWA Genesis are hand- held funduscameras suitable for use in most species of laboratory animals. However, for the ocular tissues of rat and mouse the KOWA 'rat camera' would be preferable (Rubin, personal communication 1994). Terminology Ophthalmologic terminology, to be used in systemic toxicity studies, was discussed and a list of ophthalmic lesions in the rat and mouse was created. Similar lists may be of use for other species provided that the 180 Table 3 Terminology in rat and mouse Eyelids Blepharospasm, diffuse thickening of eyelid, localized swelling of eyelid, blepharitis, lesion of eyelid Conjunctiva Conjunctival redness,conjunctival swelling Tears and exudation Dacryorrhoea/epiphora, chromodacryorrhoea, puru- lent discharge Bulbus Exophthalmos, buphthalmos, enophthalmos, micro- phthalmos, anophthalmos, panophthalmitis, phthisis bulbi Cornea (Bandshaped area of) pinpoint corneal opacities, cor- neal opacity, corneal vascularization, multifocal opaci- ties, focal corneal oedema, diffuse corneal oedema, keratitis, keratoconus, corneal defect Anterior chamber Hyphema,hypopyon Ir;s Iris coloboma, iris dystrophia, anterior synechia, pos- terior synechia, rubeosis iridis, iris swelling, iris atro- phia, ectopic pupilla, iris malformation, persistent pupillary membrane lens Nuclear lens opacity, anterior lens opacity, posterior lens opacity, (multi) focal lens opacity/opacities, total lens cataract, opacification around suture lines, ante- rior lens luxation, posterior lens luxation Vitreous body Vitreous haemorrhage, persistent hyaloid artery Fundus Hyperreflectivity, pale fundus, focal retinal atrophy, complete retinal atrophy, retinal vascular congestion, attenuated retinal vessels, diffuse retinopathy, (multi) focal circumscribed retinopathy, (multi) focal non-cir- cumscribed retinopathy, retinal folds, focal ablatio re- tinae, total ablatio retinae keywords are adapted according to the specific anatomy and disorders. With sui- table (also commercially available) software, on-line input of clinical parameters is possible. An option for entry of detailed description of the lesions or of missing keywords should be available as well. Input Kuiperet al. of localization, type and severity of lesions should also be possible. Terminology is listed in Table 3. Guidelines The guidelines (OECD, EEC, USA, Canada, Japanl are fairly brief with respect to ophthalmologic examinations. Frequency of examinations, number of animals and the structures to be examined are mentioned. The ophthalmologist is free in the choice of the equipment. The requirements to be fulfilled in accor- dance with the guidelines are listed in Tables 4-7. The guidelines are listed in Table 8. Discussion Ophthalmologic examination and histo- pathology focus on morphological changes in the eyes of laboratory animals. However, functional retinal deficits are detected best with electroretinography (ERG). This non- invasive clinical method reveals changes even before structural damage is morpholo- gically detected (Maertins et al. 1993). Even when inspection of the fundus is troubled by opalescence of the ocular media, ERG is possible. The introduction of this objective method to assess changes in retinal function is hampered by the cost of the equipment, the eleborate procedure and restrictions in risk assessment. The species and strains of laboratory animals differ in morphological and phy- siological characteristics of their ocular tissues. Variations in the shape of the lens among laboratory animal species are the result of ecological adaptations, i.e. prevail- ing light conditions. Moreover, its size is related to the size of the globe. Therefore a thorough knowledge of normal anatomy and spontaneously occurring ophthalmic disorders is required for interpretation of the changes observed during ophthalmolo- gic examination (Hayes 1985, Schiavo 19901. In addition, extrapolation of results to man may be difficult because of mor- phological and functional differences. Spontaneously occurring ophthalmic disor- ders in a number of laboratory animal Ophthalmologic examination in systemic toxicity studies 181 Table 4 Ophthalmoscopy: Guidelines for pharmaceuticals/drugs/medicinal products Number to Country/ be scored organiza- per group Groups to Recovery Type of Refer- tion Duration Admin Species Frequency and per sex be scored group exam ences USA86 CHRON R sTA-EV- 10 CTR-TOP OPHTH ERY3M-TER USA 87. SUBCHR NR-1 sTA-TER All animals All 2 USA 87 CHRON NR-1 sTA-EV- All animals All 2 ERY3M-TER Canada 90 >2 days NR-1 PRI-TER All animals All 3 Canada 90 ~ 3 mo NR-1 PRI-TER All animals All Yes 3 Japan 89 1-3 mo NR-1 PRI-DUR All animals All Yes RECENT 4 Japan 89 6-12 mo NR-1 PRI-DUR All animals All Yes RECENT 4 Japan 89 1-3 mo R DUR A fixed no. All Yes RECENT 4 of animals Japan 89 6-12 mo R DUR A fixed no. All Yes RECENT 4 of animals EEC91 REPEAT R STA-DUR-TER A fixed no. All APPROP S of animals EEC91 REPEAT NR-2 STA-DUR-TER All animals All APPROP S EEC88 3mo Oral NR-2 PRI-TER All animals CTR-TOP OPHTH 6 EEC88 3mo Oral R PRI-TER CTR-TOP OPHTH 6 EEC88 3mo DERM R PRI-TER CTR-TOP OPHTH 6 EEC88 3mo INHAL R PRI-TER CTR-TOP OPHTH 6 Country/Organization: EECEuropean Economic Community (at present: European Union), OECDOrganization for Eco- nomic Cooperation and Development Duration: CIC Chronic toxicity or carcinogenicity with chronic toxicity, sUBCHRSubchronic toxicity, CHRONChronic toxi- city, REPEATRepeat dose toxicity Administration: DERMDermal, INHAL Inhalation, - = not specified Species: RRodent, NR-' Nonrodent, NR-2 Dog or monkey, MAM-1 Two mammalian species, MAM-2 At least one mam- malian species, e.g. rat, rabbit, guineapig, MAM-3 Mammal, preferably rat Frequency: STAAt start of treatment, EVERY3M Repeat every three months, TERAt termination, PRIPrior to treatment, DURDuring treatment period Groups to be scored: CTR-TOPControl and highest dose group . Recovery: All woups and the recovery groups must be investigated Type of examination: OPHTHOphthalmoscopy, RECENTWith a recently developed ophthalmoscope, APPROPWith an appropriate device References: (seeTable 8) Table 5 Ophthalmoscopy: Guidelines for agrochemicals Groups to Recovery Type of Refer- be scored group exam ences Country/ organiza- tion Duration Admin Species Frequency USA86 CHRON R sTA-TER USA87 SUBCHR NR-1 STA-TER USA 87 CHRON NR-1 STA-TER Japan 89 3mo Diet MAM-1 PRI-TER Japan 89 3mo DERM MAM-2 PRI-TER Japan 89 3mo INHAL MAM-3 PRI-TER Japan 89 CHRON Diet R PRI-TER Japan 89 C/C Diet NR-1 PRI-TER EEC88 3mo Oral NR-1 PRI-TER EEC88 3mo Oral R PRI-TER EEC88 3mo DERM R PRI-TER EEC88 3mo INHAL R PRI-TER Abbreviations asTable 4 Number to be scored per group and per sex 10 All animals All animals All animals All animals All animals All animals All animals CTR-TOP All CTR-TOP CTR-TOP CTR-TOP CTR-TOP All CTR-TOP CTR-TOP CTR-TOP CTR-TOP OPHTH 1 2 2 7 7 7 7 7 OPHTH 6 OPHTH 6 OPHTH 6 OPHTH 6 182 Kuiper et al. Table 6 Ophthalmoscopy: Guidelines for chemicals· Number to Country/ be scored organiza- per group Groups to Recovery Type of Refer- tion Duration Admin Species Frequency and per sex be scored group exam ences USA 86 CHRON R STA-TER 10 OPHTH 1 USA 87 SUBCHR NR-l STA-TER All animals All 2 USA 87 CHRON NR-l STA-TER All animals All 2 EEC88 3 mo Oral NR-l PRI-TER All animals CTR-TOP OPHTH 6 EEC88 3 mo Oral R PRI-TER CTR-TOP OPHTH 6 EEC88 3 mo DERM R PRI-TER CTR-TOP OPHTH 6 EEC88 3 mo INHAL R PRI-TER CTR-TOP OPHTH 6 OECD81 3 mo Oral R PRI-TER All animals CTR-TOP OPHTH 8 OECD81 3 mo Oral NR-1 PRI-TER All animals CTR-TOP OPHTH 8 OEeD 81 3 mo DERM - PRI-TER All animals CTR-TOP OPHTH 8 OEeD 81 3 mo INHAL - PRI-TER All animals CTR-TOP OPHTH 8 Abbreviations asTable 4 Table 7 Ophthalmoscopy: Guidelines for food additives Number to Country/ be scored organiza- per group Groups to Recovery Type of Refer- tion Duration Admin Species Frequency and per sex be scored group exam ences UsA86 CHRON R sTA-EV- 10 CTR-TOP OPHTH 1* ERY3M-TER USA 87 sUBCHR NR-1 sTA-TER All animals All 2* USA 87 CHRON NR-1 STA-EVERY- All animals All 2* 3M-TER EEC88 3 mo Oral NR-1 PRI-TER All animals CTR-TOP OPHTH 6 EEC88 3 mo Oral R PRI-TER CTR-TOP OPHTH 6 EEC88 3 mo DERM R PRI-TER CTR-TOP OPHTH 6 EEC88 3 mo INHAL R PRI-TER CTR-TOP OPHTH 6 Abbreviations asTable 4 *USA guidelines 1986 and 1987 for food additives are also applicable for pharmaceuticals Table 8 References to Guidelines 1. Regulatory guidelines No.1, Rodent carcinogenicity and chronic toxicity, IRI, USA, (1986) 2. Regulatory guidelines No.4, Non-rodent toxicity, IRI, USA, (1987) 3. Drugs directorate guidelines, toxicologic evaluation, Health and Welfare, Canada (1990) (pp 4 and 19) 4. Notification No. 24 of the Pharmaceutical Affairs Bureau, Guidelines for toxicity studies required for applica- tions for approval to manufacture (import) drugs, Ministry of Health and Welfare, Japan, (1989) 5. The rules governing medicinal products in the European Community,Vol.l:The rules governing medicinal pro- ducts for human use inthe European Community, EEC(1991) 6. Directive 87/302, EEC,annex V of the EECDirective 67/548, EECPart B: methods for the determination of toxicity 90 day oral rodent and non-rodent toxicity, EEC(1988) (pp 9 and 13) 7. Japanese test guidelines, laws and regulations for agricultural chemicals No: II, Ministry of Agriculture, Forestry and Fisheries. Society of Agricultural Chemical Industry, (1985) 8. Guideline for testing of chemicals, Nos 408, 409, 411and 413 for rodent subchronic oral, non-rodent subchronic oral, subchronic dermal and subc:hronic inhalation, GECD (1981) Ophthalmologic examination in systemic toxicity studies species are described in literature (Taradach et al. 1992). Toxic compounds may con- tribute to a higher incidence of senile lesions, accelerate a process of ageing, influence prenatal development or provoke specific ocular changes. Acknowledgment We are grateful to Mark Vogels who took the pictures (Figs I, 2 and 31 in the animal house of Notox BV. References Abrams KL, Brooks DE, Funk RS, Theran P (1990) Evaluation of the Schirmer Tear Test in clinically normal rabbits. American Journal of Veterinary Research 51, 1912-13 Anderson GW, Lawrence WB, Lee J-O, Young M (1991) A restraint for ophthalmic examination of un-anaesthetized rats. Laboratory Animal Science 41, 288-90 Bamett KC (1990) A Colour Atlas of Veterinary Ophthalmology. London: Wolfe Publishing Ltd Bokobza MM (1988) Contribution a l'etude icono- gmphique et histologigue du segment postirieur de l'oeil chez les animaux de laboratoire (thesis). Ecole Nationale Veterinaire d'Alfort Gelatt KN (1991) Veterinary Ophthalmology. Phila- delphia: Lea and Febiger Gelatt KN, Pfeiffer RL, Erickson JL, Gum GG (19751 Evaluation of tear formation in the dog, using a modification of the Schirmer Tear Test. Journal of the American Veterinary Medical Association 166, 368-70 Hayes AW (1985) Toxicology of the eye, ear and other special senses: target organ toxicity series. New York: Raven Press Hockwin 0, et al. (1992) Manual of Oculotoxicity testing of drugs. Fischer Verlag Jaax GP, Graham RR, Rozmiarek H (1984) The Schirmer Tear Test in Rhesus monkeys (Macaca mulatta). Laboratory Animal Science 34, 293-4 Knepper PA, Collins JA, Frederick R (1995) Effect of dexamethasone, progesterone and testosterone on lOP and GAG's in the rabbit eye. Investigative Ophthalmology and Visual Science 26, 1093-100 Lee VHL (1983) Esterase activities in adult rabbit eyes. Journal of Pharmaceutical Sciences 72, 239-48 Lee P (1992) Letter to the editor. Laboratory Animal Science 42, 433 Maertins T, Kroetlinger F, Sander E, Pauluhn J, Machemer L (1993) Electroretinographic assess- ment of early retinopathy in rats. Archives of Toxicology 67, 120-5 Moore CG, Scott TM, Morrison JC 11993) Noninva- sive measurement of rat intraocular pressure with the Tono-Pen. Investigative Ophthalmology a