INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF CERTAIN
VETERINARY DRUG RESIDUES IN FOOD
WHO FOOD ADDITIVES SERIES 41
Prepared by:
The 50th meeting of the Joint FAO/WHO Expert
Committee on Food Additives (JECFA)
World Health Organization, Geneva 1998
DICLAZURIL (addendum)
First draft prepared by
Professor F.R. Ungemach
Institute of Pharmacology, Pharmacy and Toxicology
Veterinary Faculty, University of Leipzig, Leipzig, Germany
1. Explanation
2. Biological data
2.1 Toxicological studies
2.2 Toxicokinetics
2.3 Developmental toxicity
3. Comments
4. Evaluation
5. References
1. EXPLANATION
Diclazuril, an anticoccidial drug, was evaluated at the
forty-fifth meeting of the Committee (Annex 1, reference 119), when a
temporary ADI of 0-20 µg/kg bw was established on the basis of a NOEL
of 3 mg/kg bw per day in a two-year study of toxicity and
carcinogenicity in mice and a safety factor of 200. The Committee
noted a lack of toxicokinetic data showing enteric absorption of
diclazuril in the studies of teratogenicity in rabbits and concluded
that there was no evidence that the exposure of the dams to diclazuril
had been sufficient to allow evaluation of the embryotoxicity and
reproductive toxicity of the drug. The Committee requested a further
study of teratogenicity in rabbits and evidence that the doses
administered were sufficiently high for the teratogenic potential of
diclazuril to be adequately explored.
Since the previous evaluation, two pilot dose range-finding
studies including the toxicokinetics of diclazuril and a full-length
study of teratogenicity with repeated doses have been conducted in
rabbits. The additional information is summarized and discussed in
this addendum.
2. BIOLOGICAL DATA
2.1. Toxicological studies
Rabbits
In two separate studies, diclazuril was administered by oral
gavage as an aqueous suspension to groups of seven rabbits for two
weeks, in one study at doses of 0, 80, 160, or 320 mg/kg bw per day
(Dom et al., 1995) and in the other at 0, 320, 640, or 1280 mg/kg bw
per day (Dom et al., 1996). These studies were designed as dose
range-finding studies for a subsequent investigation of teratogenicity
and were not performed in compliance with GLP. The animals were
checked periodically for body weight, food consumption, clinical signs
of adverse reactions, haematological and serum parameters, organ
weights, and gross and histopathological appearance (the last in the
first study only). At day 10, blood samples were taken from three
randomly selected animals of each group for toxicokinetic analysis.
No deaths, abnormal clinical signs, or adverse effects on body
weight or food consumption were reported. Slight changes seen in some
haematological and serum parameters and in the weights of the heart
and pancreas were not dose-dependent and can be considered not to be
related to treatment. A dose-dependent decrease in the weight of the
gonads, with no gross or histopathological changes, was observed in
the first study but not at the higher doses in the second study. The
results indicate that orally administered diclazuril is well tolerated
by female rabbits at doses up to 1280 mg/kg bw per day.
2.2 Toxicokinetics
In order to demonstrate oral absorption and systemic exposure of
the animals to diclazuril during tthe two-week studies described
above, serial blood samples were collected 0, 2, 4, 12, and 24 h after
administration of the dose from three randomly selected animals of
each group at day 10 in the first study (Sterkens, 1997a) and at day
13 in the second (Sterkens, 1996). Plasma levels of diclazuril were
also determined at day 18 in the study of teratogenicity described
below in two animals per group, 1, 2, 4, 8, and 24 h after treatment
(Sterkens, 1997b). Diclazuril was analysed by a validated gas
chromatography method with electrochemical detection (limit of
quantification, 50 ng/ml). The plasma levels declined very slowly in
all studies, suggesting saturation and/or poor elimination. The
quality of the data was not such as to enable an adequate evaluation
of the Cmax or of the area under the concentration-time curve.
Table 1 shows the peak plasma levels derived by visual inspection of
the plasma concentration curves, indicating enteric absorption and
systemic exposure of female rabbits to orally administered diclazuril.
Owing to variations between animals and assays and saturation of
absorption at doses > 640 mg/kg bw, no linear dose-dependency of
the plasma levels of diclazuril could be observed.
Table 1. Peak plasma levels (Cmax, µg/ml) of orally administered
diclazuril in rabbits
Reference Dose (mg/kg bw)
80 160 320 640 1280
Sterkens (1997a) 3.7 3.8 6.40
Sterkens (1996) 6.06 8.20 7.41
Sterkens (1997b) 4.2/7.9 7.4/21.2 13.1/14.9
Values are means of three determinations
2.3 Developmental toxicity
Rabbits
In a study of developmental toxicity, groups of 18 randomly
selected pregnant albino rabbits were given an aqueous solution of
diclazuril by oral gavage at doses of 0, 80, 320, or 1280 mg/kg bw per
day on days 6-18 of pregnancy. The doses were selected on the basis of
the findings of the two-week studies and the toxicokinetic analysis
(Dom et al., 1995, 1996; Sterkens, 1996, 1997a). The does were
observed for body-weight changes, food intake, behaviour, and
mortality. At day 18 of treatment, serial blood samples were taken
from two animals of each group for analysis of the plasma levels of
diclazuril. The toxicokinetics were reported separately (Sterkens,
1997b) and are described above. Animals that aborted were killed and
necropsied; the remaining animals were killed at day 28 of pregnancy,
and the uteri, ovaries, and litters were checked for signs of
embryotoxicity and teratogenicity. The study was carried out in
compliance with GLP and according to the rules governing medicinal
products in the European Community and to the Guidelines on Detection
of Toxicity to Reproduction for Medicinal Products published by the US
Food and Drug Administration. Appropriate statistical analysis was
performed with non-parametric tests.
No compound-related mortality was observed, and diclazuril had no
adverse effect on body weight or maternal weight gain. Food
consumption was sightly reduced in all treated groups but increased
after cessation of treatment. The only clinical signs reported in
animals at the intermediate and high doses were reduced faeces
production in three and abortions in four. Uterine weights were
comparable in all groups. A slight, nonsignificant increase in the
number of total resorptions was seen at doses up to 320 mg/kg bw per
day, and the highest dose caused a significant increase in the
frequency of early and late resorptions and a slight decrease in the
number of live fetuses and in mean litter size. No substance-related
effect on preimplantation loss was observed, whereas the frequency of
post-implantation loss was increased at 1280 mg/kg bw per day. The sex
ratio and fetal weights were comparable in all groups. Examination of
fetuses for external, visceral, and skeletal anomalies revealed minor
abnormalities and variations in those at the low and intermediate
doses. At 80 mg/kg bw per day, no difference from the vehicle controls
was observed, but at 320 mg/kg bw per day an increased number of
fetuses with minor variations, such as plaques in cranial sutures,
fused sternum, and rudimentary first pair of ribs, was reported.
Malformations were detected in a few fetuses at doses < 320 mg/kg
bw per day, but these changes were considered not to be related to
treatment because of the lack of dependency on dose, their absence in
previous studies with diclazuril in rabbits at doses up to 160 mg/kg
bw per day (Annex 1, reference 119), and the appearance of similar
malforma-tions in untreated historical controls. The group at the
highest dose showed a pronounced increase in the incidence of
abnormalities and of major malformations (palatoschisis,
cheiloschisis, deformation or absence of cranial bones, meningoceles,
protrusion of brown masses in the brain, hyperossification of
vertebrae, and sternum deformations), which occurred in 48% of
fetuses, affecting 92% of the litters. These malformations were
considered to be related to treatment. Thus, high doses of diclazuril
(1280 mg/kg bw per day) were teratogenic to rabbits, whereas a dose of
320 mg/kg bw per day was not teratogenic but slightly fetotoxic. The
NOEL for developmental toxicity was 80 mg/kg bw per day (Dom et
al., 1997).
3. COMMENTS
The Committee considered additional information on the toxicity
of diclazuril in non-pregnant female rabbits and on its toxicokinetics
in pregnant and non-pregnant animals. The teratogenicity of the
compound was also investigated. The studies were carried out in
accordance with appropriate standards for study protocol and conduct.
Two separate two-week pilot studies of toxicity including
toxicokinetics were performed in female rabbits in order to define
doses for a definitive study of teratogenicity. Diclazuril was
administered by gavage to groups of seven animals at doses of 0, 80,
160, or 320 (first study) and 0, 320, 640, or 1280 mg/kg bw per day
(second study). The compound was well tolerated at all doses.
For toxicokinetic analysis, serial blood samples were collected
over 24 h from three rabbits at each dose on day 10 (first study) or
day 13 (second study). The plasma concentrations of diclazuril showed
only minor fluctuations throughout the sampling period and showed no
clear dependency on dose and no linear increase with increasing dose.
The results indicate saturable intestinal absorption of orally
administered diclazuril and demonstrate systemic exposure of the
treated animals to the test compound.
In a study of developmental toxicity, groups of 18 pregnant
rabbits were given daily doses of 0, 80, 320, or 1280 mg/kg bw
diclazuril by gavage on days 6-18 of pregnancy. All animals were
killed on day 28. Studies of toxicokinetics performed on day 18
confirmed the previous findings of saturable absorption and plasma
levels of diclazuril ranging from 4 µg/ml at the lowest dose to
15 µg/ml at the highest. Diclazuril was well tolerated by the dams at
all doses, with no deaths. The only clinical signs observed were
slightly reduced food intake during treatment, reduced faeces
production, and a few abortions at doses of 320 and 1280 mg/kg bw per
day. Examination of the litter parameters revealed no compound-related
adverse effects at doses up to 320 mg/kg bw per day, whereas the
incidences of resorption and postimplantation loss were significantly
increased at the highest dose. Examination of the fetuses revealed a
low incidence of minor compound-related abnormalities at 320 mg/kg bw
per day; at 1280 mg/kg bw per day, treatment with diclazuril
significantly increased the number of abnormalities and induced
malformations in 48% of the fetuses. The Committee concluded that
diclazuril is teratogenic in rabbits at high doses, whereas doses up
to 320 mg/kg bw per day are slightly fetotoxic but not teratogenic.
The NOEL for developmental toxicity was 80 mg/kg bw per day.
4. EVALUATION
The additional studies complied with the request of the Committee
by showing enteric absorption and exposure of pregnant rabbits to
diclazuril which was sufficient to enable the teratogenicity of the
drug to be assessed. On the basis of these findings, the Committee
considered the NOEL for diclazuril of 3 mg/kg bw per day in the
two-year study of toxicity and carcinogenicity in mice reviewed at the
forty-fifth meeting, based on histopathological changes in the lungs,
as the most appropriate toxicological end-point for establishing an
ADI. An ADI of 0-30 µg/kg bw was established by applying a 100-fold
safety factor to the NOEL. This provides a margin of safety of at
least 10 000 for the teratogenic effect of the compound in rabbits.
5. REFERENCES
Dom, P., Benze, J., Coussemont, W., Fransen, J., Lampo, A. & van
Cauteren, H. (1995) Two-week oral toxicity study in rabbits.
Unpublished report No. R064433, Exp. No. 3682 from Janssen Research
Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
Belgium.
Dom, P., Benze, J., Coussemont, W., Vandenberghe, J., Lampo, A. & van
Cauteren, H. (1996) Two-week oral toxicity study in rabbits.
Unpublished report No. R064433, Exp. No. 3762 from Janssen Research
Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
Belgium
Dom, P., Benze, J., Coussemont, W., Fransen, J., Lampo, A. & van
Cauteren, H. (1997) Oral (gavage) study for effects on embryo-foetal
development (segment II) with R064433 (Exp. No. 3373). Unpublished
report from Janssen Research Foundation. Submitted to WHO by Janssen
Pharmaceutica NV, Beerse, Belgium
Sterkens, P. (1996) Toxicokinetics of diclazuril (R064433) in the
albino rabbit in a two-week oral toxicity study (Exp. No. 3762).
Unpublished protocol No. R064433/FK 2119 from Janssen Research
Foundation. Submitted to WHO by Janssen Pharmaceutica NV, Beerse,
Belgium
Sterkens, P. (1997a) Non-clinical pharmacokinetics report: Addendum to
the toxicity report exp. No. 3682. Unpublished report No. R064433/FK
2037 from Janssen Research Foundation. Submitted to WHO by Janssen
Pharmaceutica NV, Beerse, Belgium
Sterkens, P. (1997b) Non-clinical pharmacokinetics report: Addendum to
the toxicity report exp. No. 3373. Unpublished report No. R064433/FK
2432 from Janssen Research Foundation. Submitted to WHO by Janssen
Pharmaceutica NV, Beerse, Belgium
See Also:
Toxicological Abbreviations
Diclazuril (WHO Food Additives Series 36)
DICLAZURIL (JECFA Evaluation)