Toxicology, 6 (1976) 41--46 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

THE O R A L TOXICITY OF CLIOQUINOL (5-CHLORO-7-IODO-8-HYDROXYQUINOLINE) IN BEAGLE DOGS

R. HEYWOOD, H. CHESTERMAN and A.N. WORDEN

Huntingdon Research Centre, Huntingdon PE18 6ES (Great Britain) (Received October 21st, 1975) (Accepted December 10th, 1975)

SUMMARY

When clioquinol was administered to Beagle dogs, disturbances in gait which were associated with abnormal reflexes and reactions, were seen in animals receiving 250 and 400 mg/kg b o d y weight per day. Histopathological examination of the central nervous system (CNS) showed pathological change in the posterior columns of the spinal cord.

INTRODUCTION

Halogenated oxyquinoline derivatives have been employed for many years in the treatment of non-specific diarrhoea in man. In the late fifties, Japanese neurologists started to describe a new t y p e of myelo-neuropathy, which in 1965 was recognised as a new clinical entity and called sub-acute myelooptic neuropathy, or SMON. The aetiology of SMON is not known, though the general opinion is that clioquinol intake may be implicated [1]. Attempts to reproduce SMON in experimental animals have yielded variable, and sometimes equivocal, results. Tateishi et al. [ 2] noted degeneration in the spinal cord of a dog given clioquinol. The same group [3] reported peripheral and spinal lesions in mongrel dogs, comparable to those seen in SMON patients. In a subsequent publication Tateishi et al. [4] reported apparent strain differences in the response of dogs to clioquinol. The findings of the Japanese were n o t confirmed by Hess et al. [5] or by Hess et al. [6]. A study of clioquinol was undertaken at the Huntingdon Research Centre, the results of which are reported in this communication.

Abbreviations : CNS, central nervous system; SGPT, serum glutamic-pyruvate transaminase; SMON, sub-acute myelo-optic neuropathy.

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METHODS 24 pure-bred Beagle dogs were allotted to 4 groups each of 3 males and 3 females; 3 groups received clioquinol as a powder in gelatine capsules, while the fourth group received empty gelatine capsules and acted as controls. The test compound was administered daily for 26 weeks. During the first week of the study the dose levels administered were 150, 100 and 50 mg/kg body weight/day, but from week two onwards the dose levels were increased to 400, 250 and 100 mg/kg body weight/day. The mean weight of the animals at the start of the dosing period was 6.595 kg and the mean age 17 weeks. The dogs were housed singly and were offered 400 g of Spratt's Dog Diet daily; 300 ml of milk was given daily and water was freely available. Throughout the trial, any clinical signs were noted daily, and body weight was recorded once a week. Ophthalmoscopic examinations were performed before the start of the trial period and subsequently at regular intervals. After the onset of neurological symptoms, neurological examinations based on the methods of Palmer [7] and Foster [8] were carried out at regular intervals. Routine laboratory investigations were performed before the trial period, then after 4, 8, 12, 20 and 24 weeks administration of the test compound. Haematological investigations included determination of erythrocyte sedimentation rate, haemoglobin, haematocrit, erythrocyte count, platelet count, reticulocyte count and prothrombin time. Clinical biochemistry comprised estimation of plasma glucose and urea, serum alkaline phosphatase, SGPT and total serum protein, with electrophoretic breakdown into albumin and globulin fractions. Urinalysis included determination of specific gravity and qualitative tests for protein, glucose, ketones, bile pigments, bile salts, blood pigments and microscopy of the sediment left after centrifugation of a urine sample. At the end of the test period the animals were killed and subjected to full macroscopic examination. All the major organs were weighed and pieces of tissue were removed and prepared for histological examination. Particular attention was paid to the nervous system. RESULTS 2 dogs receiving the high dose (400 mg/kg body weight/day) were killed for humane reasons before the full course of treatment had been administered. One male animal was killed on day 47 and a female animal was killed on day 161. Both animals had developed abnormal gait, with loss of use of the hind limbs and general deterioration of their condition. The male animal showed a slight increase in the SGPT level. When tissues were examined the only abnormality observed in this dog's tissues was in the liver, in which a small number of vacuolated and occasionally degenerating hepatocytes were seen. However, in the female dog killed on day 161, the fasciculus gracilis of the cervical spinal cord showed degenerative changes that were of undoubted pathological significance.

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TABLE I NEUROLOGICAL EXAMINATION

250mg/kg/day

Dosage

400 mg/kg/day

D o g No. a n d

53c~ 5 5 c ~ 5 7 c ~ 54(2

5 6 c ~ 58(2

5 9 c ~ 61c~

6 3 c ~ 60(2

62Q

64(2

sex

Postural reflex Week 4 7 13 19 24 Sensation Week

4 7 13 19 24

Segmental reflex Week 4 7 13 19 24 Gait Week

4 7 13 19 24

Pathological change -- posterior c o l u m n s o f the spinal cord

0 0 0 + 0

0 0 + + 0

0 0 ++ 0 Dead + + Dead

0 + ++ ++ ++

0 + + 0 +

0 + + 0 ++

0 + + 0 +

0 0 + 0 ++

0 + + 0 ++

0 + + + +

0 0 0 0 0

0 0 0 + 0

0 0 + + 0

0 0 + 0 Dead + 0 Dead

0 + + 0 +

0 + 0 0 0

0 0 0 0 0

0 0 0 0 0

0 + + + +

0 + + 0 +

0 0 + + +

0 0 0 0 0

0 0 0 + +

0 + + + +

0 0 + 0 Dead + 0 Dead

0 + 0 + +

0 + + 0 +

0 + + 0 0

0 + 0 0 0

0 0 + + +

0 0 0 0 +

0 0 0 + ++

0 0 0 + 0

0 0 0 ++ +

0 0 0 ++ +

+ 0 ++ 0 D e a d ++ ++ Dead

+ + ++ ++ ++

0 + ++ ++ +

0 0 + ++ +

0 0 0 0 0

0 0 0 + +

0 0 0 0 +

0 0 + 0 ++

0 0 0 0 0

+

++

0

++

+

+

0

+

+

+

0

+

O, n o r m a l ; +, a b n o r m a l ; ++, s e v e r e l y a b n o r m a l ; _+, d o u b t f u l .

Abnormal animals day.

The

mg/kg

reflex reactions

body

weight/day

in the gait was first noted

incidence

animals

and

least affected

with some splaying ed showed

abnormal

400

Unsteadiness

tration. Those

gait and

receiving

complete

timing showed limb

during

250

mg/kg

is s h o w n

a slight incoordination

paralysis.

stance.

were body

seen in weight/

week 4 of drug adminis-

of this abnormality

of the limbs to maintain hind

and responses and

The most

The condition

i n T a b l e I.

of the hind limbs severely affect-

was not progressive,

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Fig. 1. Cervical spinal cord x 200. Fasciculis gracilis showing abnormal degree of vacuolation and myelinophages are present. for o n c e a certain stage had been r e a c h e d t h e animals a d a p t e d and in s o m e cases i m p r o v e m e n t was evident. The p a t t e r n s o f reflex responses are presented in Table I. The cranial nerve reflexes were n o r m a l t h r o u g h o u t t h e s t u d y . The p o s t u r a l r e a c t i o n s w h i c h rely on the integrity o f the spinal reflex arc and the ascending and d e s c e n d i n g spirial tracts, were c o n s i s t e n t l y affected. The

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segmentary reflexes, which are dependent on the spinal reflex arc were involved, particularly towards the end of the study. Attempts were made to map areas showing lack of sensitivity to pain stimuli; three animals receiving 250 mg/kg b o d y weight/day showed negative responses to pain stimuli over the hind limbs. All dogs were gaining weight satisfactorily before the start of the trial, and there was no immediate adverse effect when the test c o m p o u n d was first administered. However, b o d y weight gain was suppressed in animals receiving 400 mg and 250 mg/kg weight/day, although f o o d and water consumption were unaffected. No adverse effects were noted in the eyes, nor were there any significant changes in the haematological and biochemical parameters monitored. After 4 weeks of drug administration serum alkaline phosphatase and SGPT levels were increased in individual dogs, receiving 400 mg/kg b o d y weight/day. Subsequently no group differences were detected between dosed and control dogs. Macroscopic post mortem examination did not reveal any significant change, and the organ weights were within normal limits. As a result of the histological examination it was found that, of those four animals receiving the highest dosage level {400 mg/kg b o d y weight/day) that survived the dosing period, all showed pathological changes in the fasciculus gracilis of the spinal cord. Similarly, 4 of the 6 animals that had received 250 mg/kg b o d y weight/day, showed some changes. Dogs receiving 100 mg/kg b o d y weight/day were unaffected. The chief morphological features of the changes were axonal degeneration and swelling, and breakdown of myelin with myelinophages (Fig. 1). The female animal that had been killed at day 161 showed also degenerative changes of the optic nerve. 3 dogs receiving 400 mg/kg b o d y weight/day and 1 dog receiving 250 mg/kg b o d y weight/day showed signs of hepatotoxicity. Histological examination revealed a small number of vacuolated and occasionally degenerating hepatocytes, which were seen mainly in the portal and some centrilobular areas, associated with mononuclear cells. There was some increase in connective tissue elements. DISCUSSION

The clinical s y m p t o m s and the pathological picture described in this paper are not at variance with those described by the Japanese workers. The repeated neurological examinations showed weekly variations in sensory m o t o r function, which hampered diagnosis and prognosis of damage to the CNS. However, the neurological s y m p t o m s described are not inconsistent with damage to the spino cerebellar tracts, which convey proprioceptive impulses from the muscles and joints to the cerebellum. The cranial nerve reflexes were normal throughout the study, and detailed ophthalmological examinations did not reveal any eye damage, although histological examination showed that the optic nerve of one dog receiving 400 mg/kg b o d y weight/day contained numerous degenerative fibres with ballooned axon

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sheaths. Tateishi et al. [4] described loss of visual accuity with vacuolation and loss of axons in the optic tract. The diagnosis of disturbance of vision in the dog is difficult in cases of toxicity in which optic nerve damage has been induced; Heywood [9] found impairment of the pupillary light reflexes to be the first clinical sign. In Sweden, Lannek and her colleagues [10--16] surveyed cases of alleged toxicity associated with the administration of halogenated oxyquinolines to dogs admitted to a veterinary clinic, and attempted to reproduce the condition in both mongrels and Beagles by dosing with clioquinol. In both spontaneous and experimental cases of alleged oral intoxication in dogs, hepatotoxicity and a focal myocardial necrosis with interstitial cellular reactions, especially in the left ventricle, were reported. Degenerative neural changes were demonstrated in the brain and spinal cord, with accompanying chromatolysis and vacuolation, especially in the hippocampus and hypothalamus. Further studies sponsored by Ciba-Geigy have been carried out at the Huntingdon Research Centre, and have confirmed and extended the findings presented in this paper. They will be published separately in due course. REFERENCES 1 R. Kono, Japan. J. Med. Sci. Biol., 24 (1971} 195. 2 J. Tateishi, H. Ikeda, A. Saito, T. Kuroda, T. Hayahara and S. Otsuki, Igaku no Ayumi, 77 (1971) 205. 3 J. Tateishi, S. Kuroda, A. Saito and S. Otsuki, Lancet, 2 (1971) 1263. 4 J. Tateishi, S. Kuroda, A. Saito and S. Otsuki, Lancet, 1 (1972) 1289. 5 R. Hess, H. Keberle, W.P. Koella, K. Schmid and J. Gelzer, Lancet, 2 (1972) 424. 6 R. Hess, W.P. Koella, G. Krinke, H. Petermann, P. Thomann and F. Zak, ArzneimittelForsch., 23 (1973) 1566. 7 A.C. Palmer, Introduction to Animal Neurology, Blackwell, Oxford, 1965. 8 S.J. Foster, J. Small Animal Pract., 9 (1968) 295. 9 R. Heywood, Br. Vet. J., 130 (1974} 564. 10 B. Lannek, Acta Vet. Scand., 14 (1973} 723. 11 B. Lannek, Acta Vet. Scand., 15 (1974) 15. 12 B. Lannek and P. Lindberg, Svensk. Vet., 11 (1972a} 438. 13 B. Lannek and P. Lindberg, Acta Vet. Scand., 13 (1972b) 441. 14 B. Lannek and P. Lindberg, Acta Vet. Scand., 15 (1974a) 398. 15 B. Lannek and P. Lindberg, Acta Vet. Scand., 15 (1974b1 419. 16 B. Lannek and L. Jonsson, Acta Vet. Scand., 15 (1974) 26.

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