Pesticide residues in food — 2007: Toxicological ... - ipcs inchem

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physically examined for changes in general health status) and quantitative assessments of landing
foot splay, muscle weakness (fore- and hindlimb grip strength) and sensory perception (tail-flick test)
were made in week −1, and in weeks 2, 5, 9 and 14. The observations were made by one observer who
was “blind” with respect to the treatment of the rats, and recorded on a computer system by personnel
not directly involved in the clinical observations. The examination proceeded from the least to the
most interactive test with observations recorded as follows:
(i) Assessment in the home cage: bizarre behaviour (circling, head-flicking, head-searching, walking
backwards, rolling-over sideways, paw-flicking), vocalization.
(ii) Removal from the cage: approach response, response to touch (increased, decreased),
v ocalization.
(iii) In the standard arena: activity (increased, decreased), comatosed, prostration, hunched posture,
bizarre behaviour (circling, head flicking, head searching, walking backwards, rolling over sideways,
paw flicking), convulsions (tonic, clonic), vocalization, ataxia, tremors, reduced stability, abnormal
gait, splayed gait, tiptoe gait, reduced limb function (fore-, hind-), upward curvature of the spine,
downward curvature of the spine, piloerection, sides pinched in, ungroomed appearance, urinary
incontinence, diarrhoea.
(iv) Handling the rat: response to touch, convulsions, vocalization, tremors, piloerection, skin
colour (pale, hyperaemia, cyanosis), ungroomed appearance, hyperthermia, hypothermia, chromodacryorrhoea,
lachrimation, ptosis, endophthalmus, exophthalmus, miosis, mydriasis, stains around
the mouth, stains around the nose, salivation, respiratory abnormalities (breathing rate, breathing
depth, laboured breathing, gasping, irregular breathing, whistling, wheezing, croaking), thin appearance,
sides pinched in, dehydration, abdominal tone (increased, decreased), urinary incontinence,
d iarrhoea.
(v) Reflex tests: righting reflex (from supine position), response to sound (to finger click/clap), splay
reflex (degree of splay when rat was lifted by base of tail), visual placing response (rat was lifted by
base of tail and slowly moved downwards towards the edge of arena), pupil response to light (after
eye had been held closed for 10 s), palpebral membrane reflex (palpebral membrane touched with
bristle and blink response observed), corneal reflex (hair was touched against cornea and blink reflex
observed (only performed if palpebral reflex was absent), pinna reflex (bristle poked into ear canal),
foot-withdrawal reflex (to toe pinch).
(vi) Quantitative measures: forelimb and hindlimb grip strength, landing-foot splay, time to t ail‐flick.
Locomotor activity was monitored by an automated activity recording apparatus (Coulbourn
Lab Linc Infra-red Motion Activity System), which records small and large movements as an activity
count. Rats were placed individually in stainless steel cages with an infra-red sensor attached and the
recording session started immediately. All rats were tested in weeks −1, 2, 5, 9 and 14 of the study.
Each observation period was divided into ten scans of 5 min duration, during which the rats did not
have access to food, water or items of environmental enrichment. Treatment groups were counterbalanced
across test times and across devices, and when the trials were repeated each rat was returned
to the same activity monitor at approximately the same time. At the end of the dosing period, five
males and five females per group were killed, then fixed by perfusion in situ and subjected to neuropathological
examinations. The liver was removed and weighed from non-perfused rats from the
control group and the group at 2000 mg/kg bw per day. The remaining rats were killed under carbon
dioxide anesthaesia without any further examinations. The stability, homogeneity and correctness of
the concentrations of difenoconazole in the diet were analytically verified.
There were no difenoconazole-related adverse effects at any dose. Body weight was lower than
that of controls for both sexes at 1500 ppm for all or most of the study (maximum difference, 10%
and 6% respectively). There was no effect of treatment on food consumption at any dose. Food utilization
for males at 1500 ppm was lower than that of controls throughout the study. Hindlimb grip
strength was lower for males at 1500 ppm in weeks 9 and 14. Since this pattern was observed at the
DIFENOCONAZOLE 201–272 JMPR 2007