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    TRANQUILLIZERS

         The tranquillizing agents considered by the Committee have
    several points in common. All are old compounds in their class, for
    all of them the data available to the Committee were inadequate in
    certain respects, all are often used shortly before slaughter in
    pigs, and all leave residues in edible tissues. Furthermore,
    administration by injection is known to create a local area of high
    concentration of the drug which, in part, is likely to be present at
    slaughter and, if in edible tissue, is a potential hazard to the
    consumer.

         The Committee therefore advises against the use of these drugs
    for any purpose in the immediate pre-slaughter period, especially
    when given by injection into the tissues.

    AZAPERONE

    First Draft prepared by
    Dr. G. Roberts, Environmental Health Branch
    Department of Community Services and Health
    Canberra, Australia

    1.  EXPLANATION

         Azaperone is a butyrophenone neuroleptic tranquillizer for use
    in pigs. The therapeutic dose range is 0.4 to 2.0 mg/kg given
    intramuscularly. This is the first occasion on which azaperone has
    been evaluated by the Joint FAO/WHO Expert Committee on Food
    Additives.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

         Rats were given an oral dose of 1 mg/kg tritiated azaperone in
    aqueous 0.01 M tartaric acid. Over a 4-day period the amount of
    radioactivity recovered was 16% in urine and 81% in faeces, most of
    which was collected within the first 24 hours. At the end of the
    4-day period, less than 1% of the dose was found in organs and
    tissues, with highest levels in the liver, kidney and heart
    (Heykants, 1973).

         After a subcutaneous injection of 1 mg/kg azaperone, rats
    excreted 20-25% in urine, most within 24 hours, and 60-80% in faeces
    mostly within 48 hours. At the end of 4 days, radioactivity could
    not be detected in tissues (Heykants et al., 1971b).

         In subcutaneously dosed rats, peak levels of total
    radioactivity and unchanged azaperone were detected in blood, liver
    and brain within 30 min. Thereafter, elimination of azaperone from
    brain and blood was rapid (down to 1% of peak after 8 hours) but
    slower in liver (down to 25% of peak after 8 hours). Total
    radioactivity diminished more slowly indicating slower elimination
    of metabolites (Heykants et al., 1971a).

         The fate of a subcutaneous injection of azaperone was similar
    in pregnant rats. Peak levels in placenta and fetus occurred after
    60 min followed by rapid elimination. In tissues, the proportion of
    radioactivity as unchanged drug decreased quickly indicating rapid
    degradation of azaperone and slower elimination of metabolites
    (Heykants, 1974).

         Azaperol, a metabolite of azaperone, was given by intravenous
    injection to rats. Measurement of liver, kidney and brain tissue
    levels revealed elimination half-lives of 45 min, 15 min and 15 min
    respectively. Some 6% of the dose was converted to azaperone (Rauws
    et al., 1976).

    2.1.2  Biotransformation

         After an oral dose in rats, only 1.5% of urinary radioactivity
    and 34% of faecal radioactivity were in the form of unchanged drug.
    By comparison in subcutaneously injected rats, less unchanged
    azaperone (12%) was found in faeces (Heykants, 1973).

         The biotransformation of azaperone was rapid in subcutaneously
    dosed rats and was thought to occur mainly in the liver. As early as
    15 min post treatment, 75% of radioactivity in the liver was in the
    form of metabolites (Heykants et al., 1971a).

         The excreta from subcutaneously dosed rats were analyzed for
    degradation products. The main metabolites arose from oxidative
    removal of the pyridyl group (3 in Fig. 1) and acetylation of the
    resulting free piperazine. The former, which was almost exclusively
    in faeces, the latter was in both urine and faeces and totalled
    about 50% of radioactivity. Another three metabolites, amounting to
    15% of the dose and representing oxidative N-dealkylation, were
    found in both urine and faeces (Heykants et al., 1971b).

         Using rat liver fractions  in vitro, azaperone was shown to be
    metabolized to a greater extent by the 16000 x g supernatant than
    the microsomal fraction. The main metabolic pathways were reduction
    of the butanone (2 in Fig. 1), hydroxylation of the pyridine group
    (7 in Fig. 1), oxidative N-dearylation (3 in Fig. 1) and oxidative
    N-dealkylation (Meuldermans et al., 1973).

         Azaperone was incubated with the 16000 x g supernatant from rat
    liver for 1 hour at 37C. About 10% was unmetabolised, 22% was as
    azaperol (2 in Fig 1), 15% was due to hydroxylation of the pyridine
    group (7 in Fig 1) with lesser amounts of metabolites 3, 4, 8, 9,
    and 10 in Fig 1 (Meuldermans et al., 1975).

    2.1.3  Effects on hepatic enzymes

         Mice were injected subcutaneously with 4 mg/kg azaperone. At
    the end of 7 days, the protein content of liver or microsomes was
    unaffected. Cytochrome P-450 concentration was increased but
    NADPH-cytochrome C-reductase activity was reduced (Pekkanen and
    Salminen, 1973).

    FIGURE 1

    2.2  Toxicological studies

    2.2.1  Acute studies

        Table 1. Results of acute studies with azaperone
                                                                                  

    Species        Sex        Route            LD50          Reference
                                             (mg/kg b.w.)
                                                                                  

    Mouse           M         oral             385           Niemegeers et
                    M         s.c.             179           al., (1974)
                    M         i.v.             38-42

    Rat             M         oral             245
                    M         s.c.             450
                    M         i.v.             28

    Guinea          M         oral             202
    pig

    Dogs           N.S.       oral            > 20
                   N.S.       s.c.            > 20
                                                                                  
    
         The major toxic signs in rodents were ptosis, sedation, tremors
    and occasionally clonic seizures. Ptosis and sedation were observed
    in dogs with vomiting after oral dosing.

         In mice, the i.v. LD50 values for metabolites 2 and 8 were 56
    and 150 mg/kg respectively, higher than for azaperone (Niemegeers,
    1975).

    2.2.2  Short-term studies

    2.2.2.1  Rats

         Groups of 10 male and 10 female Wistar rats were given
    subcutaneous injections of 0, 2.5, 10, and 40 mg/kg b.w./day
    azaperone for 13 weeks. The vehicle used was 0.9% NaCl while the
    purity of azaperone was not stated. 

         Treated rats in all groups were sedated for 2 hours following
    dosing and at 40 mg/kg b.w./day passive behaviour was exhibited
    throughout the study. There were no deaths due to treatment. In
    males only, body weight gain was non-significantly reduced at 2.5
    and 10 mg/kg b.w./day and markedly reduced at 40 mg/kg b.w./day.

         Haematology, blood chemistry and urinalysis were examined at
    the end of the study. The only apparent effects were a shift in the
    differential white cell count from lymphocytes to neutrophils in
    males at 10 mg/kg b.w./day and males and females at 40 mg/kg
    b.w./day and a slight increase in serum alkaline phosphatase in
    males at 10 and 40 mg/kg b.w./day.

         At necropsy, spleens "looked degenerated" at 40 mg/kg b.w./day.
    Thymus weights were decreased in males at 10 mg/kg b.w./day and
    males and females at 40 mg/kg b.w./day. In females, liver weights
    were increased and ovaries showed reduced numbers of corpora lutea
    and increased glandular tissue at 40 mg/kg b.w./day (Marsboom et
    al., 1967).

         Groups of 10 male and 10 female Wistar rats were fed diets
    containing 0, 100, 400, or 1600 ppm azaperone (purity 98-102%) for
    15 weeks. There were no signs of toxicity during the study. Food
    consumption and body weight gain were depressed at 1600 ppm.
    Ophthalmoscopy, haematology, blood chemistry and urinalysis were
    examined at the end of the study. The only notable findings were
    decreased serum cholesterol in males and females, increased
    urobilinogen in males and increased urinary creatinine in females,
    all at 1600 ppm.

         Gross pathology was unremarkable. Brain weights were heavier at
    1600 ppm. Slight bile duct proliferation was seen in livers of 400
    and 1600 ppm males. In females, ovaries showed "active large corpora
    lutea" and there were "reduced eosinophils" in the uterine wall,
    "mucified aspect" of vaginal mucosa, more developed alveolar tissue
    in the mammary gland and "stimulation of erythrosinophils" in the
    pituitary. The effects in females were expressed at 1600 ppm and to
    a lesser extent at 400 ppm. The NOEL was 100 ppm, equal to 10 mg/kg
    b.w./day (Marsboom et al., 1969).

         Groups of 10 male and 10 female Wistar rats were fed diets
    containing 0, 100, 400, or 1600 ppm azaperone (purity not stated)
    for 6 and 12 months. Based on food intake the average doses were 8,
    31, and 130 mg/kg b.w./day for 6 months and 8, 30, and 127 mg/kg
    b.w./day for 12 months.

         Dose-related sedation was observed in all drug-treated groups
    during the entire experimental period. Survival was unaffected. Food
    consumption and body weight gains were depressed at 1600 ppm while
    in the 400 ppm group weight gain was affected in the 6-month study
    only.

         Ophthalmoscopy, haematology, blood chemistry and urinalysis
    were examined at the end of 6 and 12 months. Serum cholesterol was
    decreased in the 1600 ppm group at 6 months but not at later times.
    Serum bilirubin, BUN and urinary urobilinogen were higher in females
    of the 1600 ppm group at 6 and 12 months.

         At autopsy, gross pathology was unaffected. Brain weight was
    increased at 1600 ppm after both 6 and 12 months of dosing. Septal
    cell proliferation in the lung was marked at 6 and 12 months in the
    1600 ppm group and led to lipoid pneumonia.


         The females of the 1600 ppm group exhibited a "prolonged
    diestrus in the uterus" (atrophic at 12 months) accompanied by
    "reduced activity in the ovaries" (low numbers of active corpora
    lutea and abundant interstitial glandular tissue), "mucification and
    thin layered epithelium" with no cornification in the vagina and
    "more extensive chromophobe tissue" in the pituitary. These findings
    in reproductive tissues were more marked at 12 months. Apart from
    the pharmacological effect of sedation, the NOEL was 8 mg/kg
    b.w./day (Marsboom et al., 1976a).

    2.2.2.2  Dogs

         Groups of 3 male and 3 female beagle dogs were given 0, 1.25,
    5, or 20 mg/kg b.w./day of azaperone (purity 99.7%) for 13 weeks.
    Dosing was carried out during 6 days per week in capsules.

         Dogs of the 20 mg/kg b.w./day group exhibited a sedative effect
    for 3 to 4 hours post-dosing in addition to decreased general
    activity, ptosis and catatonia. Emesis and salivation were seen
    occasionally at 5 mg/kg b.w./day and frequently at 20 mg/kg
    b.w./day. Physical examinations revealed one female in each drug
    treatment group with transient swelling of mammary glands.

         Body weight was not clearly affected by dosing. Ophthalmoscopy,
    ECG, blood pressure, haematology, blood biochemistry and urinalysis
    were examined pre-treatment and monthly during the study. None of
    these parameters was altered.

         At terminal necropsy, liver weights tended to increased values
    at 5 and 20 mg/kg b.w./day, but a dose-relationship was not evident.
    Gross and histopathology were unaffected. The NOEL was 1.25 mg/kg
    b.w./day (Marsboom et al., 1973).

    2.2.3  Long term/carcinogenicity studies

    2.2.3.1  Rats

         Concomitant with the 6 and 12 month rat studies (see Section
    2.2.2.1), groups of 10 male and 10 female Wistar rats were fed diets
    containing 0, 100, 400, or 1600 ppm azaperone (purity not stated)
    for 18 months. Based on food intake the average doses were 7, 29,
    and 115 mg/kg b.w./day for 18 months.

         As in the shorter-term studies, sedation was noted at all doses
    while food intake and weight gain were depressed at 1600 ppm.
    Ophthalmoscopy, haematology, blood biochemistry and urinalysis were
    examined at the end of the study. Serum bilirubin and BUN and
    urinary urobilinogen were increased in females of the 1600 ppm
    group.

         At necropsy, brain weight was increased in 1600 ppm rats. Gross
    pathology was unremarkable. Septal cell proliferation in the lung
    leading to lipoid pneumonia was marked at 1600 ppm. Effects seen in
    the pituitary, ovary, uterus, and vagina at 6 and 12 months were not
    evident in this 18-month experiment. Tumours were not increased.
    Pharmacological effects were noted at all doses, the NOEL for
    toxicological effects was 29 mg/kg b.w./day (Marsboom et al.,
    1976a).

    2.2.3.2  Dogs

         Groups of 3 male and 3 female beagle dogs were given 0, 1.25,
    5, and 20 mg/kg b.w./day of azaperone (purity not stated) for 24
    months. Animals were dosed 6 days/week in capsules.

         One male dog given 20 mg/kg b.w./day azaperone died in the 64th
    week. Signs of intoxication included sedation, back arching,
    protrusion of the tongue, head shaking, muscle tremors, apnea,
    lacrimation, increased salivation, and emesis. These effects were
    seen in most dogs at 20 mg/kg b.w./day and some dogs at 5 mg/kg
    b.w./day with sporadic emesis and salivation at 1.25 mg/kg b.w./day.
    Body weight gain was unaffected.

         Ophthalmoscopy, ECG, BP, haematology, blood biochemistry and
    urinalysis were examined pre-dosing and every 3 months during the
    study. None of these parameters was altered.

         At autopsy, there was increased bile on the duodenal mucosa in
    the 5 and 20 mg/kg b.w./day groups. The weights of adrenal glands
    and the liver were increased at 20 mg/kg b.w./day. The following
    microscopic changes were noted mainly in females of the 1.25 and
    5 mg/kg b.w./day groups with virtually no effects at 20 mg/kg
    b.w./day: "more marked or protracted metoestral period" -- active
     corpora lutea in 2 low-dose (LD) and 1 mid-dose (MD), "fatty
    superficial epithelium" in uterus of 2 LD and 2 MD, "more resting
    aspect" of genital tract with "thin layered vaginal epithelium" in
    all LD and 2 MD, atrophy of uterine wall in 1 high-dose (HD),
    stimulation of mammary gland in LD and MD and "stimulation of
    erythrosinophilic tissue" in the pituitary in 2 LD. A NOEL was not
    identified (Marsboom et al., 1976b).

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         A 3-generation study was carried out in Wistar rats. The
    initial generation (F0) was allowed to deliver and suckle pups.
    The second generation (F1) was mated within treatment groups
    avoiding brother-sister matings, and was allowed to deliver and
    suckle pups. The third generation (F2) was mated as in the F1
    but the females were killed on gestation day 22.

         Azaperone (purity not stated) was administered in the diet at
    concentrations of 0, 25, 100, and 400 ppm food. The dietary levels
    resulted in doses of approximately 2.5, 10, and 40 mg/kg b.w./day.
    Only adult females were treated, on gestation days 6 to 15 in each
    generation. Group sizes were 20 F0 females, 29-33 F1 females,
    and 40-52 F2 females.

         There were no mortalities among treated dams in any generation
    but other toxic signs were not recorded. Maternal weight gain and
    pregnancy rate were similar between groups.

         Litter size, pup weight and post-natal weight gain were
    unaffected in F1 and F2 offspring. Pup survival was reduced at
    40 mg/kg b.w./day during the F2 lactation period only.

         Uterine examination of F2 dams revealed no effects on
    implantation, resorption or fetal weight. In the 40 mg/kg b.w./day
    group, there were 2 F3 fetuses without metacarpal bones of the
    foreleg and 1 without metatarsal bones of the hind leg (unilateral).
    The NOEL was 10 mg/kg b.w./day (Marsboom, 1974a).

    2.2.5  Special studies on embryotoxicity and teratogenicity

    2.2.5.1  Mice

         Groups of 29 pregnant CRL:COBS-CD-1 mice were given gavage
    doses of 0, vehicle, 2.5, 10, or 40 mg/kg b.w./day of azaperone
    (purity not stated). Treatment was on gestation days 6 to 15 and
    females were killed on gestation day 18. The vehicle contained
    tartaric acid, sodium bisulfite, methyl and propyl paraben.

         A number of animals died in the vehicle and drug-treated
    groups, death was attributed to dosing difficulties as a result of
    conditioned aversion to the vehicle. Decreased activity, ptosis,
    impaired righting reflex and catalepsy were seen 1 to 3 hours
    post-dosing with 10 and 40 mg/kg b.w./day azaperone.

         Maternal weight gain was reduced in the vehicle control group
    and further reduced at 10 and 40 mg/kg b.w./day. This effect on body
    weight may be a reflection of lower litter sizes which were due to
    both reduced numbers of corpora lutea and slightly increased
    resorptions. Implantation rate, fetal weight and survival were not
    significantly affected.

         The ratio of male to female fetuses was reduced in the vehicle
    and 40 mg/kg b.w./day groups. Fetal examination revealed a slight
    delay in ossification of tarsals and phalanges of fore and hind paws
    at 40 mg/kg b.w./day. Gross and visceral abnormalities were not
    induced. The NOEL was 2.5 mg/kg b.w./day (Mosher et al., 1973).

    2.2.5.2  Rats

         Groups of pregnant Wistar rats were given gavage doses of 0,
    2.5, 10, or 40 mg/kg b.w./day of azaperone (purity not stated) in an
    aqueous vehicle. Treatment was on gestation days 6 to 15 and females
    were killed on gestation day 22.

         Maternal survival and weight gain were unaffected. The number
    of implantations and live and dead fetuses was similar in all
    groups. Fetal weight and gross, visceral, and skeletal examinations
    were unremarkable. There was no effect at the highest dose of
    40 mg/kg b.w./day (Marsboom, 1972a).

         Groups of 20 pregnant Wistar rats were given subcutaneous doses
    of 0, 2.5, 10, or 40 mg/kg b.w./day of azaperone (purity not stated)
    in an aqueous vehicle. Treatment was on gestation days 6 to 15 and
    females were killed on gestation day 22.

         There was no mortality among the dams but weight gain was lower
    in the 10 and 40 mg/kg b.w./day groups. The number of implantations
    was similar in all groups. Relative to the number of implantations,
    the number of resorptions was slightly increased at 40 mg/kg
    b.w./day, resulting in slightly smaller litter size. Fetal weight
    was reduced at 40 mg/kg b.w./day and the only fetal abnormality was
    scoliosis in one 40 mg/kg b.w./day fetus (Marsboom, 1973a).

         Groups of 20 pregnant Wistar rats were given subcutaneous doses
    of 0, 2.5, 10, or 40 mg/kg b.w./day of azaperone (purity not stated)
    in an aqueous vehicle. Treatment was on gestation days 1 to 21 and
    females were killed on gestation day 22.

         There was no maternal mortality. Weight gain in the dams was
    reduced at 10 and 40 mg/kg b.w./day. The number of implantations was
    lower at 10 and 40 mg/kg b.w./day and resorptions were increased at
    40 mg/kg b.w./day. Fetal weight was depressed at 40 mg/kg b.w./day
    but there was no increase in fetal abnormalities (Marsboom, 1967).

         Groups of 25 pregnant Wistar rats were given gavage doses of 0,
    2.5, 10, or 40 mg/kg b.w./day of azaperone (purity not stated) in an
    aqueous vehicle. Treatment was from gestation day 16 to post-partum
    day 21 and dams were allowed to deliver naturally and nurse young
    throughout lactation.

         There were no maternal deaths and the duration of gestation and
    parturition were unaffected. Body weight gain of dams in the 2.5 and
    40 mg/kg b.w./day groups was reduced. Litter size, birth weight, and
    post-natal weight gain were unremarkable. The survival of pups
    through the lactation period was compromised at 40 mg/kg b.w./day.
    There were no fetal abnormalities. The NOEL was 10 mg/kg b.w./day
    (Marsboom, 1973b).

    2.2.5.3  Golden hamsters

         Groups of 26 pregnant golden hamsters were given gavage doses
    of 0, vehicle, 2.5, 10, or 40 mg/kg b.w./day of azaperone (purity
    not stated). Treatment was on gestation days 6 to 10 and females
    were killed on gestation day 15. The vehicle contained tartaric
    acid, sodium bisulfite, methyl and propyl paraben.

         There was no maternal mortality. Toxic signs were ptosis from
    2.5 mg/kg b.w./day, decreased motor activity from 10 mg/kg b.w./day,
    catalepsy and impaired righting reflex at 40 mg/kg b.w./day during
    the 2 hours following dosing.

         Body weight gain in dams was depressed at 40 mg/kg b.w./day,
    and was associated with lower fetal weight at this dose. The number
    of implantations, resorptions and litter size was similar between
    groups. Fetal examination revealed only a slight delay in
    ossification of metatarsals at 40 mg/kg b.w./day. Discounting
    pharmacological effects, the NOEL was 10 mg/kg b.w./day (Mosher et
    al., 1974).

    2.2.5.4  Rabbits

         Groups of 15 pregnant NZ White rabbits were given gavage doses
    of 0, 2.5, 10, or 40 mg/kg b.w./day of azaperone (purity not stated)
    in an aqueous vehicle. Treatment was on gestation days 6 to 18 and
    females were killed on gestation day 28.

         There were no maternal deaths and body weight gain was
    depressed at 10 and 40 mg/kg b.w./day. Resorptions or fetal death
    were not induced but litter size was slightly lower in the 40 mg/kg
    b.w./day group, reflecting lower numbers of implantations. There
    were no fetal abnormalities related to treatment. The NOEL was
    2.5 mg/kg b.w./day (Marsboom, 1972b).

    2.2.6  Special studies on genotoxicity

        Table 2. Results of genotoxicity studies on azaperone1
                                                                                               

    Test system     Test object         Concentration        Results       Reference
                                                                                               

    Ames test2      S.typhimurium

                    TA 1538             > 750 g/plate       positive3     Preiss et al.,
                    TA 1537             (+ S9 mix)                         1982, 1983
                    TA 98
                    TA 1535             2500 g/plate        negative      Scheutwinkel-
                    TA 100              (+ S9 mix)                         Reich, et al.,
                                                                           1982

                    TA 1535             2500 g/plate
                    TA 1537             (- S9 mix)
                    TA 98
                    TA 1538
                    TA 100

    Ames test2      S.typhimurium

                    TA 1530             2000 g/plate4       negative      Poncelet et al.,
                    TA 1535             (+ & - S9                          1982; Duvergervan
                    TA 1537             mix)                               Bogaert et
                    TA 1538                                                al., 1987
                    TA 989
                    TA 100

    Micro           Rats                20-160 mg/kg         negative      Vanparys &
    nucleus test                        orally                             Marsboom, 1982

    Dominant        Mice                10-160 mg/kg         negative      Marsboom,
    lethal test                         orally                             1974b
                                                                                               

    1. Appropriate positive controls were used.
    2. Both with and without rat liver S9 fraction.
    3. There was no dose relationship.
    4. Higher concentrations were bacteriotoxic.

    Table 3. Results of Ames tests on azaperone metabolites1,2
             (Scheutwinkel-Reich et al., 1982)
                                                                                               
    Compound                           Strain           Concentration              Results
                                                                                               
    alpha-(4-fluorophenyl)-4-          TA 98            > 500 g/plate (+ S9)      positive2
    (2-pyridinyl)-piperazine
    butanol (Azaperol)

                                       TA 1538          1500 g/plate (+ S9)       positive

                                       TA 1535          2500 g/plate              negative
                                       TA 1537          (+ S9)
                                       TA 100

                                       TA 98            2500 g/plate              negative
                                       TA 1538          (- S9)
                                       TA 1535
                                       TA 1537
                                       TA 100

    4-(4-acetyl)-1-piperazinyl-        TA 1538          5000 g/plate              positive
    41-fluoro-butyrophenone                             (+ S9)

                                       TA 98            5000 g/plate              negative
                                       TA 1535          (- S9)
                                       TA 1537
                                       TA 100

                                       TA 98            5000 g/plate              negative
                                       TA 1538          (+ S9)
                                       TA 1535
                                       TA 1537
                                       TA 100

    -(p-fluorobenzoyl)-               TA 98            2500 g/plate              positive2
    propanoic acid                                      (+ S9)

                                       TA 1538          5000 g/plate              negative
                                       TA 1535          (- S9)
                                       TA 1537
                                       TA 100

    p-fluorobenzoyl                    TA 98            5000 g/plate              negative
    acetic acid                        TA 1538          (+ and - S9)
                                       TA 1535
                                       TA 1537
                                       TA 100
                                                                                               
    1. Appropriate positive controls were used throughout
    2. Response not dose-related
    
    2.2.7  Special studies on pharmacology

        Table 4. Results of studies with azaperone in pharmacological tests ( Niemegeers
             et al, 1974).
                                                                                               

                                       ED50 value          ED50 value         ED50 value
                                       (mg/kg SC)          (mg/kg SC)         (mg/kg SC)
                                                                                               

    Type of test                         Rat                Mouse               Dog
                                                                                               

    amphetamine antagonism              2.5                  -                   -

    apomorphine antagonism              0.34/9.15            -                  0.98

    norepineprine antagonism            0.33                 -                   -

    tryptamine antagonism               5.9                  -                   -

    jumping box test                    0.7                  -                 5(3.95 OR)

    W-test
    - body weight                       1.75                 -                   -
    - food intake                       2.5                  -                   -
    - faecal output                     4.0                  -                   -

    behavioural observations
    - catalepsy                         8.0                  -                   -
    - ptosis                            1.5                  -                   -

    open field test
    - ambulation                        6.7                  -                   -
    - rearing                           4.1                  -                   -
    - defecation                        6.9                  -                   -

    traumatic shock test                0.021                -                   -

    thermoregulation                    3.27                 -                   -
    - 37 C                             > 320                -
    - 30 C

                                                                                               

    Table 4. cont'd
                                                                                               

                                       ED50 value          ED50 value         ED50 value
                                       (mg/kg SC)          (mg/kg SC)         (mg/kg SC)
                                                                                               

    Type of test                         Rat                Mouse               Dog
                                                                                               

    tail withdrawal test                > 40                 -                   -

    inhibition of food intake           > 10                 -                   -

    hot plate test                       -                   7.0                 -

    inhibition of righting reflex        -                   > 40                -

    pentobarbital potentiation           -                   0.4                 -

    rotating rod test                    -                   1.64                -

    fighting test                        -                   0.74                -
                                                                                               

    1. lowest effective dose, the NOEL was 0.01 mg/kg sc.
    
    2.3  Observations in humans

         A group of 20 male psychotics were studied; 10 remained on their 
    previous medication, 10 had their medication replaced with azaperone. 
    Doses were commenced at 0.5 mg three times daily (t.i.d.) increasing 
    to 20 mg t.i.d over a 17-day period.  The maximum dose was then 
    administered for 2 months.

         Clinical observation revealed no symptoms up to 2 mg t.i.d. of 
    azaperone. At higher doses (from 2.5 mg t.i.d.), sedation was observed
    in a dose-related manner and at 20 mg t.i.d. patients started to 
    complain of dizziness. Haematology and blood chemistry parameters, 
    examined prior to and at the end of 2 months azaperone treatment, were 
    within the normal range (Reyntjens, 1972).

    3.  COMMENTS

         A range of studies on azaperone was submitted for assessment
    including data on kinetics and metabolism, acute toxicity,
    short-term and long-term toxicity, developmental toxicity, and
    genotoxicity. Most of the studies were carried out in the 1970s and
    the standard of testing and reporting varied widely.

         The kinetic studies with azaperone were insufficient to
    determine the extent of absorption from the gastrointestinal tract.
    However, by comparison with the excretion pattern after parenteral
    dosing, it was estimated that absorption after oral dosing was
    probably high. Distribution within the body in rats was extensive
    and excretion was primarily in the faeces (81%), with lesser amounts
    in urine (16%). Azaperone is extensively and rapidly metabolized.
    Two metabolites were found in the pig but not in the rat. However,
    these compounds are devoid of significant pharmacological activity,
    and therefore do not affect the suitability of the rat as a model
    for toxicological testing.

         Azaperone was moderately toxic in acute toxicity studies in
    mice, rats, guinea pigs, and dogs. Most signs of intoxication
    reflected exaggerated pharmacological activity of azaperone in the
    central nervous system. A battery of pharmacological studies
    indicated that azaperone possesses potent anti-alpha-adrenergic
    activity, but these data were inadequate for use in determining a
    NOEL as the drug was almost always given by the subcutaneous route.

         Short- and long-term toxicity studies were carried out in rats
    and dogs. Dose-related sedation was the major effect in both species
    and was observed at all treatment levels. Minor hepatotoxicity was
    observed at doses at and above 30 mg/kg b.w./day in rats and 5 mg/kg
    b.w./day in dogs. In rats only, brain weight was consistently
    increased at 30 mg/kg b.w./day, but in the absence of any
    pathological change this observation could not be explained.

         There were pathological changes in the pituitary and sex
    organs, particularly in rats; these were typical of neuroleptic
    agents. It has been postulated that the primary effect is
    pharmacological and is caused by the blocking of dopamine receptors
    in the hypothalamus or pituitary, resulting in increased prolactin
    and decreased gonadotrophin secretion. While this could account for
    the observed slight stimulation of the pituitary and mammary glands
    and the quiescence of the female reproductive tract, direct evidence
    for this mechanism was lacking. The effects on the reproductive
    organs were slight, in line with the relatively weak anti-dopamine
    activity of azaperone. In dogs, such effects were observed only
    after dosing for 24 months with 1.25 and 5 mg/kg b.w./day but not at
    20 mg/kg b.w./day. In rats, effects were noted after 3, 6, and 12
    months but not after 18 months, which suggests the possibility of
    adaptation. When pharmacological effects were excluded, the NOELs
    were 1.25 mg/kg b.w./day in dogs and 8 mg/kg b.w./day in rats.

         The Committee noted that the carcinogenic potential of
    azaperone had not been adequately investigated. The only study in
    which lifetime exposure was approached was an 18 month study in
    rats. However, the duration of dosing was too short and the small
    group size (only ten rats of each sex) was inadequate to determine
    treatment-related tumour incidences satisfactorily.

         Frame shift mutations in  Salmonella typhimurium strains were
    seen for azaperone and three metabolites in a series of studies
    carried out by one group of investigators. However, reversion rates
    were only 2-3 times those in controls; there was no dose-response
    relationship, and high doses in the presence of rat liver microsomes
    were required. This weak response was not reproduced by a second
    group of investigators using the same bacterial strains.
    Genotoxicity was absent in the micronucleus and dominant lethal
    tests  in vivo, suggesting that azaperone has low potential for
    genetic damage.

         In a three-generation study in rats, survival of the pups was
    reduced during lactation in one generation at the highest dose of
    40 mg/kg b.w./day. There were no adverse effects on other
    reproduction parameters. It was recognized, however, that an
    unconventional methodology was used in this study in that males were
    left untreated and females were dosed on gestation days 6-15 only.
    The study was considered to be inadequate to enable the potential
    for effects on reproduction and fertility to be fully assessed.

         Embryotoxicity and teratogenicity were examined in mice, rats,
    golden hamsters, and rabbits. Fetal abnormalities were not observed
    in any species. Administration of azaperone during the gestation
    period resulted in embryotoxicity in mice at or above 10 mg/kg
    b.w./day and in rats at 40 mg/kg b.w./day. Maternal toxicity and
    fetotoxicity, in the form of delayed ossification of metatarsals and
    metacarpals in mice and golden hamsters and reduced fetal weight in
    rats, were noted at 40 mg/kg b.w./day. In a perinatal and postnatal
    study in rats, the survival of pups during the lactation period was
    reduced at 40 mg/kg b.w./day.

         Human psychotic patients treated with up to 2 mg of azaperone
    three times a day (about 0.1 mg/kg b.w./day) showed no clinical
    effects. At doses of 2.5 mg given three times daily (about
    0.125 mg/kg b.w./day) and above there was dose-related sedation, and
    at 20 mg three times daily (about 1 mg/kg b.w./day), dizziness.
    Haematological and blood chemistry parameters were not affected at
    any dose.

         There were no effects apart from sedation at 1.25 mg/kg
    b.w./day in a 24-month dog study and at 8 mg/kg b.w./day in an
    18-month rat study. There was no NOEL for pharmacological activity
    in the animals used in the toxicological studies. However, the study
    in human subjects provided additional information. The NOEL for
    sedation was 2 mg given three times a day. Since the human subjects
    were given azaperone in divided doses and it is unclear whether the
    doses were additive over the course of the day, the NOEL for
    sedation in humans was taken to be about 0.03 mg/kg b.w.

    4.  EVALUATION

         In view of the absence of adequate carcinogenicity and
    reproduction studies and the weak mutagenicity findings in bacteria,
    the Committee could not establish an ADI. The Committee was aware of
    data on the tumorigenic potential of other butyrophenone neuroleptic
    agents, but considered that the structural differences between them
    were sufficient to preclude the use of this information to support a
    temporary ADI for azaperone.

    5.  REFERENCES

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    HEYKANTS, J. (1973) The excretion and metabolism of azaperone in the
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    HEYKANTS, J. (1974) The transplacental passage of azaperone and its
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    HEYKANTS, J., LEWI, P. & JANSSEN, P.A.J. (1971a) On the distribution
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    MARSBOOM, R. (1972a) Potential of oral R1929 for embryotoxicity and
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    MARSBOOM, R. (1973a) Potential of subcutaneous R1929 for
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    MARSBOOM, R., VANDESTEENE, R., HERIN, V. & VAN BELLE, H. (1976b)
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    MEULDERMANS, W.E.G., LAUWERS, W.F.J., KNAEPS, A.G., MICHIELS, L.J.M.
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    MOSHER, A.H., DANILOVITZ, M.S. & STEELMAN, R.L. (1973) Teratology
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    MOSHER, A.H., DANILOVITZ, M.S. & STEELMAN, R.L. (1974) Teratology
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    PEKKANEN, T.J. & SALMINEN, K. (1973)  Azaperone and the hepatic
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    REYNTJENS, A. (1972) Safety evaluation of azaperone treatment.
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    by Janssen.


    See Also:
       Toxicological Abbreviations
       Azaperone (WHO Food Additives Series 34)
       Azaperone (WHO Food Additives Series 41)
       AZAPERONE (JECFA Evaluation)