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    ANTIPROTOZOAL AGENT

    DICLAZURIL

    First draft prepared by
    Dr L.T. Mulligan
    Division of Toxicology and Environmental Sciences
    Center for Veterinary Medicine
    Food and Drug Administration
    Rockville, MD, USA

    Explanation

    Biological data
         Biochemical aspects
         Absorption, distribution, and excretion

    Toxicological studies
         Acute toxicity studies
         Short-term toxicity studies
         Long-term toxicity/carcinogenicity studies
         Reproductive toxicity studies
         Special studies on teratogenicity/embryotoxicity
         Special studies on genotoxicity
         Observations in humans
         Microbiological properties
         Pharmacological effects

    Comments

    Evaluation

    References

    1.  EXPLANATION

         Diclazuril is an anticoccidial drug used in major poultry
    species, such as broiler chickens, replacement pullets and turkeys, as
    well as in rabbits, lambs and minor edible bird species. It has not
    previously been reviewed by the Committee.

         The chemical name of the compound is: 2,6-dichloro-a-
    (4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2(3H)-yl)
    benzeneacetonitrile. The structure is shown in Figure 1.

    Figure 1. Diclazuril

    CHEMICAL STRUCTURE 6

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

    2.1.1.1  Rats

         Male Wistar rats received 10 mg 14C-diclazuril/kg bw in an
    aqueous suspension. On day 1, 90% of the dosed radioactivity was
    excreted in the faeces. After 4 days, 92% was excreted in the faeces
    and 0.04% in the urine. Unchanged diclazuril (UD) accounted for most
    of the total radioactivity in the faeces, with two metabolites
    accounting for less than 0.5% (Mannens  et al., 1992).

         In another study, concentrations of total radioactivity and UD
    were measured in blood, plasma and tissues for up to 96 h after oral
    administration of 10 mg 14C-diclazuril/kg bw in an aqueous
    suspension to male Wistar rats. The absorption of diclazuril was
    limited. Most of the dose was recovered from the GI contents. Maximum
    plasma concentrations of total radioactivity and UD were approximately
    1 g/ml and occurred after 8 h. On day 1, total radioactivity in
    plasma was almost exclusively UD, but later the UD/total radioactivity
    ratio gradually declined. The blood-to-plasma ratio of total
    radioactivity was about 0.7, indicating a limited distribution to
    blood cells. Distribution to the systemic tissues was rapid but
    limited. The concentration of total radioactivity in the liver was
    about 50% of the plasma level and kidney, lung and heart levels were
    about 20-30%, while muscle and brain concentrations were 5-7% of the
    plasma levels. UD depleted from tissues with a half-life of 1.5 days.
    The half-life of total radioactivity was 53.0 h (Van Beijsterveldt
     et al., 1992).

    2.1.1.2  Rabbits

         Absorption, distribution, metabolism and excretion was studied in
    2 separate studies, with male rabbits receiving a single oral dose of
    14C-diclazuril at 1 mg/kg bw. The excretion of radioactivity was
    fairly rapid since within 48 h, 70% was recovered in the faeces and 3%
    in the urine. After 10 days, more than 98% was excreted. Gall bladder
    contained radioactivity, but accounting at most for 0.02% of the dose,
    implying that biliary excretion was a minor elimination pathway.
    Various metabolites were identified in urine. Two main metabolites
    were a glucuronide and a sulfate conjugate. In faeces, UD represented
    the main part of the radioactivity, accounting for approximately 66%
    of the dose. Some minor metabolites were observed. None of the urinary
    or faecal metabolites accounted for more than 2% of the dose. UD
    represented almost all the plasma radioactivity at least up to 120 h
    after dosing. The elimination from plasma proceeded with an apparent

    half-life of 2-2.5 days. The distribution to tissues was limited and
    the elimination half-lives were similar to that from plasma, except
    for the liver with an elimination half-life of 3 days. The easily
    extractable liver residue indicated freedom from concern over bound
    residues (Meuldermans  et al., 1988a; Michiels  et al., 1988).

    2.1.1.3  Chickens

         In a single-dose study, 14C-diclazuril was administered to
    broiler chickens in a lactose mixture at a dose of 1 mg/kg bw. Maximum
    radioactivity concentrations of 1.5-2.0 g-eq./ml were attained in
    plasma 6 h after administration. The elimination subsequently
    proceeded with a half-life of approximately 50 h.

         There was a rapid equilibrium between plasma and tissue levels.
    The tissue concentrations were 2 to 10 times lower than the
    corresponding plasma concentrations. The liver and the kidneys showed
    the highest concentrations at all time points. The half-life in
    tissues was approximately 50 h, similar to that found in plasma.
    Parent diclazuril accounted for more than 90% in the liver at 24-h
    liver, and no metabolites were detectable (<4%). About half of the
    dose was excreted within 24 h, almost exclusively as UD. After 10
    days, the cumulative excretion was over 95%. A degradation product,
    coded DM5, accounted for 5.3% of the dose in the 0-96 h excreta. Other
    metabolites accounted for <2% each. DM5 was not definitively
    identified. It was demonstrated to be a derivative of
    4-amino-2,6-dichloro-a-(4-chlorophenyl) benzeneacetonitrile, formed by
    cleavage and subsequent degradation of the triazine-dione ring
    (Meuldermans  et al., 1988b).

    2.1.1.4  Turkeys

         In a single-dose study, 14C-diclazuril was administered in a
    lactose mixture at a dose of 1 mg/kg bw. Maximum radioactivity
    concentrations of 1.78  0.19 g-eq./ml were attained in plasma 6 h
    after administration. The subsequent elimination proceeded with a
    half-life of approximately 38 h. The blood-to-plasma ratio was on
    average 0.66 throughout the 10-day observation period, implying a
    minor distribution to the blood cells. There was a rapid but limited
    distribution between plasma and tissues. The tissue concentrations
    were slightly to markedly lower than the corresponding plasma
    concentrations. The depletion rate was similar in all tissues with a
    half-life in the range of 34-46 h. UD accounted for 98% of the sample
    radioactivity for the livers sampled after 6 h, and 85% for the livers
    sampled after 48 and 72 h. No metabolites accounted for more than 10%
    of the radioactivity in liver. At 24 h after dosing, 55% of the dose
    was excreted. The cumulative excretion was 88% after 5 days and 94.8 
    0.8% after 10 days. At least 8 metabolites were identified in excreta.

    Parent diclazuril, however, accounted for the major part of the
    excreted radioactivity, representing 55.8% of the dose. The
    triazine-dione ring cleavage product previously seen in broilers
    accounted for 6.3% of the dose in the 0-96 h excreta. An unidentified
    metabolite accounted for about 2.4% of the radioactivity. All other
    metabolites accounted for < 2% each (Meuldermans  et al., 1990).

    2.1.1.5  Sheep

         Poor absorption of diclazuril was observed in sheep after oral
    administration of 1 mg/kg bw. Maximum concentrations in plasma of
    0.012 to 0.016 g/ml were reached at 24-48 h after dosing and were
    lower than the quantification limit (< 0.01 g/ml) at all other
    sampling times (Monbaliu  et al., 1993).

         In summary, in all species investigated, there were little
    indications of metabolism of diclazuril. As a result,
    diclazuril-derived substances were eliminated mainly as unchanged
    parent substance in the excreta of bird species, and in the faeces of
    rats and rabbits. Urine of the latter species was only a very minor
    excretion route and some metabolized substances could be observed. In
    edible tissues, the residues consisted almost entirely of unchanged
    diclazuril.

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with diclazuril are
    summarized in Table 1.

         Only in mice and rats dosed i.p. at 5000 mg/kg bw was mortality
    observed. Clinical effects in mice and rats were non-specific and were
    mainly on the CNS. In dogs, vomiting and defecation were seen after
    treatment. Autopsy did not reveal drug-related macroscopic changes in
    any study.

    2.2.2  Short-term toxicity studies

    2.2.2.1  Mice

         In a 3-month dose-range-finding study, SPF Albino Swiss mice
    (10/sex/group) were dosed orally at 200, 400, 800 or 1600 mg/kg of
    feed, equivalent to 30, 60, 120 or 240 mg/kg bw/day. Increased
    centrilobular swelling of the liver was observed in male mice at the
    three highest doses (3/10, 4/10, 9/10, 10/10, and 10/10 at 0, 30, 60,
    120 and 240 mg/kg bw/day, respectively), and at 240 mg/kg bw/day in
    females. At 240 mg/kg bw/day, decreased body-weight gain, increased
    liver weight and paleness of the liver were observed in males. The
    NOEL in this study was 30 mg/kg bw/day (Verstraeten  et al., 1990).

    Table 1.  Acute toxicity studies on diclazuril
                                                                        

    Species     Route      Sex         LD50            Reference
                                       (mg/kg bw)
                                                                        

    Mouse       oral       M & F       >5000           Niemegeers, 1986a

                sc         M & F       >5000           Niemegeers, 1987a

                ip         M & F       >5000           Niemegeers, 1987b

    Rat         oral       M & F       >5000           Niemegeers, 1986b

                sc         M & F       >5000           Niemegeers, 1987c

                ip         M           5000            Niemegeers, 1987d

                           F           >5000           Niemegeers, 1987d

    Rabbit      dermal     M & F       >4000           Teuns & Marsboom
                                                       1988a,b

    Dog         oral       M & F       >5000           Niemegeers, 1987e
                                                                        

         In a 3-month toxicity study, SPF Albino Swiss mice (20/sex/group)
    were dosed with diclazuril at 1000, 2000 or 3000 mg/kg of feed (equal
    to 290, 500 or 850 mg/kg bw/day in males and 290, 610 or 920 mg/kg
    bw/day in females). No mortality or adverse gross clinical effects
    were noted. At all dose levels, the males showed decreased serum total
    bilirubin and other altered serum variables, increased liver weight
    and centrilobular hepatocellular swelling. Cytoplasmic vacuolation was
    seen in 1 male at 500 mg/kg bw/day, and in 3 males at 850 mg/kg
    bw/day. A NOEL was not identified in this study. It was noted that
    feed wasting had probably caused overestimation of test article intake
    and that the calculated dosage was about twice that obtainable from
    dietary intake conversion tables (Verstraeten  et al., 1992a).

    2.2.2.2  Rats

         In two dietary studies, Wistar rats (20/sex/group) were dosed
    with diclazuril for 3 months at 50, 200 or 800 mg/kg of feed (equal to
    4, 17 or 69 mg/kg bw/day in males and 6, 21 or 89 mg/kg bw/day in
    females), and at levels of 1000, 2000 or 3000 mg/kg (equal to 71, 140
    or 210 mg/kg bw/day in males and 82, 160 or 240 mg/kg bw/day in
    females).

         In the first study, male rats dosed at 17 and 69 mg/kg bw/day
    showed centrilobular swelling with an increased presence of lipid
    vacuoles in the centrilobular hepatocytes. Increased absolute and
    relative liver weights were seen in both males and females at the
    highest dose levels. The NOELs for these liver effects were 4 and
    21 mg/kg bw/day for males and females, respectively (Verstraeten
     et al., 1986a).

         In the second study, swelling of the centrilobular hepatocytes
    was seen in both male and female groups at all doses. Hepatocytic
    cytoplasmic vacuolization was noted in males at doses of 140 and
    210 mg/kg bw/day. No mortalities occurred in either sex at any dose
    level in this study, nor were any relevant changes revealed by
    clinical chemistry, haematology or urinalysis. Relative liver weights
    were, however, increased in males and females in the highest dose
    group. A NOEL was not identified in this study (Verstraeten  et al.,
    1992b).

         In a 12-month study in Wistar rats (20/sex/group) with
    diclazuril, doses were 16, 63, 250 and 1000 mg/kg of feed (equal to 1,
    4, 18 or 74 mg/kg bw/day in males and 2, 6, 23 or 88 mg/kg bw/day in
    females). No drug-related changes were observed at doses up to 6 mg/kg
    bw/day for females and 18 mg/kg bw/day for males. Dosing at 23 and
    88 mg/kg bw/day in females resulted in the presence of histiocytic
    aggregates in the mesenteric lymph node. At 74 and 88 mg/kg bw/day in
    males and females, respectively, histological examination showed
    centrilobular swelling in the hepatocytes (males only), histiocytic
    aggregates in the mesenteric lymph node and an increased incidence of
    foamy cells in the lungs. There was no effect on clinical signs, food
    consumption, body-weight gain, mortality, haematology, serum analysis
    or urinalysis. The NOELs for the liver effects observed in this study
    were 6 mg/kg bw/day for females and 18 mg/kg bw//day for males
    (Verstraeten  et al., 1988a).

    2.2.2.3  Dogs

         Diclazuril in gelatin capsules was administered to beagle dogs
    (4/sex/group) at doses of 5, 20 or 80 mg/kg bw/day for 3 months. Two
    additional male and female animals in the control and high-dose groups
    were included for a 1-month withdrawal study. At 80 mg/kg bw/day in
    both males and females, a fine granular, yellow to brown pigment was
    seen in the cytoplasm of the hepatocytes. In males at this dose level,
    a significant increase in serum BUN values was also seen. Both liver
    pathology and BUN values were normal in the high-dose animals
    following the 1-month recovery period (Verstaeten  et al., 1986b).

         A 12-month toxicity study in beagle dogs of the same design and
    dose levels confirmed the findings of the earlier study. The liver
    changes were comparable in nature and intensity in both studies. The
    NOEL was 20 mg/kg bw/day (Verstraeten  et al., 1988b).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Mice

         In a 25-month combined long-term toxicity/carcinogenicity study,
    Swiss mice (50/sex/group) were administered doses of diclazuril of 16,
    63, 250 or 1000 mg/kg of feed (equal to 3, 11, 47 or 190 mg/kg bw/day
    in males and 4, 14, 53 or 220 mg/kg bw/day in females).

         Males at 185 mg/kg bw/day showed reduced body-weight gain and
    food consumption and an increased incidence of cachexia. Increased
    incidences of hepatic changes, mainly characterized by parenchymal
    centrilobular swelling, and fatty overload characterized by the
    presence of lipid vacuoles in the centrilobular hepatocytes and
    swollen sinusoidal cells, were seen in males at 11, 47 and 190 mg/kg
    bw/day, and in females at 220 mg/kg bw/day. The NOEL in this study was
    16 mg/kg of feed, equal to 3 mg/kg bw/day (Verstraeten  et al., 1989a).

    2.2.3.2  Rats

         Diclazuril was given in the diet for 28 months to Wistar rats
    (50/sex/group) at dose levels of 16, 63, 250 or 1000 mg/kg of feed,
    equal to 1, 4, 15 or 61 mg/kg bw/day in males and 1, 5, 20, or
    80 mg/kg bw/day in females. At 250 and 1000 mg/kg of feed,
    diclazuril-related changes were the increased presence of
    histiocytosis (males at 61 mg/kg bw/day, females at 20 and 80 mg/kg
    bw/day) and of pigmented macrophages (females at 80 mg/kg bw/day) in
    the mesenteric lymph nodes. The lung histiocytosis was observed and
    illustrated by an increased presence of foamy cells. The Committee
    regarded the highest dose used in this study to be sufficient to
    adequately assess carcinogenicity. Taking this together with the
    absence of a biologically significant increase in the occurrence of
    tumours of any type at any location, the Committee concluded that
    diclazuril was not carcinogenic in this study. The NOEL was 63 mg/kg
    of feed, equal to 4 mg/kg bw/day (Verstraeten  et al., 1989b).

    2.2.4  Reproductive toxicity studies

    2.2.4.1  Rats

         A 2-generation study was performed in Wistar rats, with each
    generation producing 2 litters. Doses were 50, 200 or 800 mg/kg of
    feed, equivalent to 5, 20 or 80 mg/kg bw/day, given continuously in
    the diet. The copulation and fertility indices and the duration of
    gestation were comparable between controls and dosed animals.
    Teratogenicity was not observed in any litter. In the first
    generation, the only adverse effect observed was a lowered birth

    weight in pups at 80 mg/kg bw/day. In the second generation,
    body-weight gain during pregnancy was slightly lower at 80 mg/kg
    bw/day, and food consumption during pregnancy and lactation was
    decreased at 20 and 80 mg/kg bw/day, indicating maternal toxicity at
    these doses. Birth weight at 80 mg/kg bw/day and the weight of pups at
    weaning and survival rate at 20 and 80 mg/kg bw/day were decreased.
    The NOEL in this study was 50 mg/kg of feed, equivalent to 5 mg/kg
    bw/day (Dirkx  et al., 1988a).

    2.2.5  Special studies on teratogenicity/embryotoxicity

    2.2.5.1  Rats

         In two studies in Wistar rats, diclazuril was given in the diet
    at 12.5-1600 mg/kg, equivalent to 1-160 mg/kg bw/day, from days 6 to
    16 of pregnancy. Dosing at 1 and 5 mg/kg bw/day did not adversely
    influence dams or their progeny. At 20-160 mg/kg bw/day, slight
    maternal toxicity was observed, characterized by a lower body-weight
    gain. Litter weights were also decreased in these dosage groups.
    Teratogenicity was not evident at any dose level. The NOEL in these
    studies was 5 mg/kg bw/day (Dirkx  et al., 1987a,b).

    2.2.5.2  Rabbits

         Oral dosing of New Zealand white rabbits with diclazuril by
    gavage from days 6 to 18 of pregnancy, at doses of 5, 20 or 80 mg/kg
    bw/day did not result in any adverse effect on the dams or their
    litter. No teratogenicity was observed (Dirkx  et al., 1988b)

         In a repeat study, doses were 40, 80 or 160 mg/kg bw/day. There
    was no evidence in these studies that the rabbits were exposed to
    sufficient diclazuril to evaluate the teratogenicity of the drug
    (Gillardin  et al., 1989).

    2.2.6  Special studies on genotoxicity

         The results of the genotoxicity studies with diclazuril are
    summarized in Table 2.

    2.3  Observations in humans

         Humans have had limited exposure to diclazuril. Since it is
    active against  Eimeria species, which is the cause of coccidiosis in
    animals, it has been administered to AIDS patients with  Isospora
     belli infection. Eight patients with  I. belli diarrhea were
    treated with 200 mg diclazuril for 7 days. Clinical improvement was
    seen and the diarrhea disappeared. No side effects of the drug was
    cited (Kayembe  et al., 1989).

        Table 2.  Results of genotoxicity studies on Diclazuril
                                                                                                                           

    Test System          Test Object                         Dose                 Results           Reference
                                                                                                                           

    DNA Repair Test      Induction of UDS in primary         0.3-30 g/ml         Negative          Weterings, 1985
                         rat hepatocytes

    SOS Chromotest       Induction of galactosidase          1-1000 ng/well       Negative S9      Vanparijs, 1985
                         synthesis in genetically
                         engineered E. coli K-12

    Ames Test            Histidine reversion in S.           10-500 g/plate      Negative          de Meester &
    (2 test reports)     typhimurium; tryptophan                                                    Leonard, 1985;
                         reversion in E. coli                                                       de Meester &
                                                                                                    Leonard, 1986

    Mouse Lymphoma       Mammalian cell gene mutation;       5-100 g/ml          Negative S9      Young, 1989
    TK+/- Assay          thymidine kinase locus;
                         trifluorothymidine resistance

    Drosophila SLRL      Sex-linked recessive lethality      500, 2000 ppm        Negative          Vanparijs, 1986a
    Test

    Human Lymphocyte     Structural chromosome               25-300 g            Negative          Leonard, 1988
    Chromosome           aberrations
    Aberrations
                                                                                                                           

    Table 2.  Results of genotoxicity studies on Diclazuril (cont'd)
                                                                                                                           

    Test System          Test Object                         Dose                 Results           Reference
                                                                                                                           

    Micronucleus Test    Induction of micronuclei in         80-5120 mg/kg        Negative          Vanparijs, 1986c;
    (2 test reports)     mouse bone marrow                                                          Vanparijs, 1991

    Mouse Dominant       Induction of dominant lethal        40-160 mg/kg         Inadequate1       Vanparijs, 1986b
    Lethal Test          mutations in male mouse germ
                         cells
                                                                                                                           

    1    No mortality or clinical signs of toxicity and no evidence of target tissue exposure to the test article were
         reported. The doses were inadequate in this test.
             Diclazuril at a similar dose was administered for crypto-
    sporidiosis in a single AIDS patient. A complete eradication of
    cryptosporidial oocysts from the faeces was seen following treatment.
    No cutaneous reactions or modification of biochemical parameter were
    documented. There were no comments on additional effects following
    drug treatment (Menichetti  et al., 1991).

    2.4  Microbiological properties

         The  in vitro antimicrobial activity of diclazuril was
    investigated in 11 pathogenic and saprogenic fungi, 6 zoo-pathogenic
    and 5 plant-pathogenic bacteria, including both Gram-positive and
    Gram-negative organisms. It was found that diclazuril possesses
    negligible antifungal and no antibacterial activity at 100 g/ml
    (Van Cutsem  et al., 1985).

         In another experiment, the antimicrobial activity of diclazuril
    was evaluated in 2 strains of  Bacillus subtilis and in 1 strain of
     Sarcina lutea, which are commonly used for testing residues of
    antibiotics in meat or other food products. No antibacterial activity
    was found (Van Cutsem  et al., 1986).

    2.5  Pharmacological effects

         In a general screen for drug activity, diclazuril was dosed to
    rats by the i.p. route at 40 mg/kg bw. Diclazuril was devoid of
    neuroleptic, sedating, analgesic, hypnotic, cholimimetic, constipating
    or anticonvulsant properties (Lampo  et al., 1995).

    3.  COMMENTS

         The Committee considered data from studies on acute, short-term,
    and long-term toxicity/carcinogenicity, reproductive and developmental
    toxicity, metabolism, pharmacokinetics and antimicrobial effects.

         Pharmacokinetic studies in rats given radiolabelled drug
    suggested that the absorption of diclazuril from the gut was limited,
    as 90% of the radioactive dose was excreted in the faeces within 24 h.
    After 4 days, 92% of the dose had been excreted in the faeces and
    0.04% in the urine. Unchanged diclazuril accounted for most of the
    total radioactivity in the faeces, with two metabolites accounting for
    less than 0.5%. Distribution into the tissues was rapid but limited.
    The concentration of total radioactivity in the liver was about half
    that in the plasma; the corresponding figures for kidney, lung and
    heart were in the range 20-30%. With time, metabolites gradually
    accounted for a higher proportion of the tissue radioactivity.

         Single oral doses of diclazuril of up to 5 g/kg bw caused no
    mortality in experimental animals. Clinical effects in mice and rats
    were non-specific and mainly of CNS origin. In dogs, vomiting and
    defaecation were seen after treatment.

         In a 3-month dose-ranging study in mice, mortality was not seen
    with oral doses up to 1600 mg/kg in the diet, equivalent to 240 mg/kg
    bw/day. At this dose, changes indicative of mild liver damage were
    reported, including increases in relative liver weight, swelling of
    the centrilobular hepatocytes and vacuolization of hepatocytes in
    males; females showed swelling of the centrilobular hepatocytes only.
    The NOEL was 30 mg/kg bw/day.

         In a 3-month toxicity study in mice in which the highest dose was
    3000 mg/kg in the diet, equal to 850 mg/kg bw/day in males and
    920 mg/kg bw/day in females, neither mortality nor adverse clinical
    effects were noted. No mortality occurred in treated mice, but at all
    dose levels (290-850 mg/kg bw/day) males showed decreased serum levels
    of total bilirubin. This and other changes revealed by haematological
    and biochemical changes in the blood, although occasionally
    statistically significant, were either marginal with respect to
    historical controls or not dose-related. Liver weights were increased
    at all doses in males and at the highest dose in females. Swelling of
    the centrilobular hepatocytes was observed in males dosed at 500 and
    850 mg/kg bw/day. A NOEL was not identified in this study. It was
    noted that wastage of feed had probably caused overestimation of
    dietary intake of the drug and that the calculated dosage was about
    twice that derived from dietary intake conversion tables.

         Rats were given diclazuril at doses of 50-800 mg/kg in the diet,
    equal to 4-69 mg/kg bw/day in males and 6-89 mg/kg bw/day in females
    for 3 months. Male rats dosed at 17 and 69 mg/kg bw/day showed
    swelling of the centrilobular hepatocytes with an increase in lipid
    vacuolization. Absolute and relative liver weights were increased in
    both males and females at the highest dose. These changes fell within
    the range of historical controls. The NOELs in this study were 4 and
    21 mg/kg bw/day for males and females, respectively.

         In a second 3-month study, in which rats were dosed at 1000-3000
    mg/kg in the diet, equal to 71-210 mg/kg bw/day in males and 82-240
    mg/kg bw/day in females, swelling of centrilobular hepatocytes
    was seen in both sexes at all doses. Vacuolization of the cytoplasm of
    hepatocytes was noted in males dosed at 140 mg/kg bw/day and above. No
    deaths occurred in either sex at any dose level in this study, nor was
    any relevant change revealed by clinical chemical analysis,
    haematology or urinalysis. Relative liver weights were, however,
    increased in males and females in the highest-dose group. A NOEL was
    not identified in this study.

         In a 12-month study in rats given diclazuril at doses of
    16-1000 mg/kg in the diet, equal to 1-74 mg/kg bw/day in males and
    2-88 mg/kg bw/day in females, no drug-related changes were observed at
    doses at or below 6 and 18 mg/kg bw/day for females and males,
    respectively. Histological examination showed histocytic aggregates in
    the mesenteric lymph node in males at 74 mg/kg bw/day and at both 23
    and 88 mg/kg bw/day in females. Swelling of centrilobular hepatocytes
    was seen in males dosed at 74 mg/kg bw/day, while females dosed at
    88 mg/kg bw/day had increased clusters of foamy cells in the lungs.
    The NOEL in this study was 6 mg/kg bw/day.

         Diclazuril capsules were administered to 4 dogs of each sex at
    doses of 5, 20 or 80 mg/kg bw/day for 3 months. Two additional male
    and female animals were included in the control and highest-dose
    groups in a 1-month withdrawal study. At 80 mg/kg bw/day in both males
    and females, a free granular, yellow brown pigment was present in the
    cytoplasm of hepatocytes. In males at this dose level, a significant
    increase in serum urea (expressed as nitrogen) was also seen. Both
    changes were reversible, as shown by the 4 animals of the recovery
    group. A 12-month toxicity study in dogs of the same design and dose
    levels as the 3-month study but lacking a recovery group, confirmed
    the findings of the earlier study. The liver changes were comparable
    in nature and intensity in both studies. The NOEL in these studies was
    20 mg/kg bw/day.

         A 25-month combined long-term toxicity/carcinogenicity study was
    performed in mice. This study included periodic haematological
    examinations, analysis of serum samples at the end of the study and
    extensive histopathological examinations. Diclazuril was administered

    at dose levels of 16, 63, 250 or 1000 mg/kg in the diet, equal to 3,
    11, 47 or 190 mg/kg bw/day in males and 4, 14, 53 or 220 mg/kg bw/day
    in females. While there was evidence of inhibition of the rate of
    body-weight gain in male mice at the highest-dose, histopathological
    examination revealed only minor hepatic morphological changes of the
    type also found in the 3-month studies in mice. In view of the results
    of the two 3-month studies in mice of the same strain, the Committee
    considered that the toxic potential of doses exceeding those used in
    the carcinogenicity study had been evaluated. Analysis of serum
    samples collected at the end of these two 3-month studies provided
    evidence of the existence of a threshold of between 1000 and
    2000 mg/kg in the diet, above which the mouse is unable to absorb
    ingested diclazuril. This threshold is close to the highest dose used
    in the long-term toxicity/carcinogenicity study. In the light of this
    finding, together with the absence of any biologically significant
    increase in the incidence of tumours, the Committee concluded that
    diclazuril had been adequately tested and was not carcinogenic in
    mice. The NOEL in this study was 3 mg/kg bw/day.

         A 28-month combined long-term toxicity/carcinogenicity study was
    performed in rats given 16, 63, 250 or 1000 mg/kg diclazuril in the
    feed (equal to 1, 4, 15 or 61 mg/kg bw/day in males and 1, 5, 20 or
    80 mg/kg bw/day in females). This study included periodic
    haematological examinations, analysis of serum samples at the end of
    the study and extensive histopathological examinations. As in the
    study with mice, no effects of obvious toxicological significance were
    noted, but reactive histiocytosis of the mesenteric lymph nodes was
    observed in female rats at 20 and 80 mg/kg bw/day and in males at
    61 mg/kg bw/day. However, as with the studies in mice, serum samples
    collected at the end of the two 3-month studies allowed an exploration
    of the relationship between the oral intake of diclazuril and the
    resultant concentration in serum. A threshold existed for the
    absorption of diclazuril in the region of 2000 mg/kg in the feed, i.e.
    about two times higher than the highest dose used in the long-term
    toxicity/carcinogenicity study. In the light of this finding together
    with the absence of any biologically significant increase in the
    incidence of tumours, the Committee concluded that diclazuril had been
    adequately tested and was not carcinogenic in the rat. The NOEL in
    this study was 4 mg/kg bw/day.

         A two-generation reproductive toxicity study was performed in
    rats in which each generation produced two litters. The doses were
    equivalent to 5, 20 or 80 mg/kg bw/day. In the first generation, the
    only adverse effect observed was a reduction in birth weight of pups
    at 80 mg/kg bw/day. In the second generation, body-weight gain of dams
    during pregnancy was slightly lower at 80 mg/kg bw/day, and food
    consumption during pregnancy and lactation was decreased at 20 and
    80 mg/kg bw/day, indicating maternal toxicity at these doses. As in
    the first generation, birth weights were lower at 80 mg/kg bw/day, and
    the survival rates and weight of pups at weaning were also decreased
    at 20 and 80 mg/kg bw/day. The NOEL in this study was 5 mg/kg bw/day.

         In two teratogenicity studies in rats, diclazuril was
    administered in the feed at doses equivalent to 1-160 mg/kg bw/day
    from days 6 to 16 of pregnancy. Dosing equivalent to 1 and 5 mg/kg
    bw/day did not result in any adverse effects on the dams or their
    progeny. At a dose equivalent to 20 mg/kg bw/day, however, slight
    maternal toxicity was observed, characterized by a reduction in
    body-weight gain. Litter weights were decreased in groups receiving
    20-160 mg/kg bw/day. At none of the doses was teratogenicity evident.
    The NOEL in these studies was 5 mg/kg bw/day.

         In two studies in rabbits, administration of diclazuril by gavage
    at doses of 5-160 mg/kg bw/day from days 6 to 18 of pregnancy did not
    result in any adverse effects on the dams or their offspring. From
    these findings and because studies of the enteric absorption of
    diclazuril in the rabbit were lacking, the Committee concluded that
    there was no evidence that exposure to diclazuril was sufficient to
    enable the teratogenicity of the drug to be evaluated. This conclusion
    was supported by the occurrence of adverse effects in rabbits exposed
    to  Eimeria spp. following administration of the drug in the feed at
    a dose equivalent to 13 mg/kg bw/day for 5 weeks. Furthermore, the
    Committee noted that materno- and fetotoxicity had been observed in
    rabbits dosed with a diclazuril analogue at 80 mg/kg bw/day.

         Diclazuril was studied in a range of  in vivo and  in vitro 
    assays with a variety of genetic end-points. Negative findings in all
    these assays enabled the Committee to conclude that diclazuril was not
    genotoxic.

    4.  EVALUATION

         The Committee established a temporary ADI of 0-20 g/kg bw for
    diclazuril, based on the NOEL of 3 mg/kg bw/day in the 2-year
    toxicity/carcinogenicity study in mice and a safety factor of 200. The
    ADI was rounded to one significant figure, consistent with accepted
    rounding procedures (Annex 1, reference 91, section 2.7).

         The results of a teratogenicity study in rabbits supported by
    evidence that the doses administered were sufficiently high for the
    teratogenic potential of diclazuril to be adequately explored are
    required for evaluation in 1998.

    5.  REFERENCES

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    See Also:
       Toxicological Abbreviations
       Diclazuril (WHO Food Additives Series 41)
       DICLAZURIL (JECFA Evaluation)