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    RACTOPAMINE

    First draft prepared by
    Dr L. Ritter
    Bureau of Veterinary Drugs
    Health Protection Branch
    Health and Welfare Canada, Ottawa

    1.  EXPLANATION

         Ractopamine is a phenolethanolamine -adrenoceptor agonist that
    is used for the improvement of weight gain, carcass leanness and
    feed efficiency in pigs. Ractopamine is marketed as the
    hydrochloride with a minimal purity of 92% and exists in two
    diastereomeric forms which are identified as RS, SR and RR, SS. The
    RR-isomer, butopamine, is a potent cardiotonic in humans.
    Ractopamine hydrochloride had not been previously 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

         Studies with 14C-ractopamine in several animal species
    indicated a rapid absorption following oral administration. Peak
    plasma and whole blood levels occurred in rats 0.5-2.0 h after
    dosing. Peak plasma and whole blood levels were similar in males
    when compared to females over the dose range of 0.5 to 2 mg/kg bw.
    At a dose of 20 mg/kg bw the levels were, however, approximately 2.5
    times higher in females than in males. The elimination half-life was
    approximately 7 h for males and females. Males receiving 20 mg/kg bw
    had an unexplained longer elimination half-life of approximately 15
    h (Dalidowicz  et al., 1986a; Williams  et al., 1987).

         Oral gavage doses of 0.05, 0.5, or 5 mg/kg bw resulted in peak
    levels in dogs 1-2 h after dosing (except for females at 5 mg/kg bw,
    when maximum concentration was reached 4-8 h after dosing). The
    elimination half-life from plasma and whole blood was approximately
    6 h (Dalidowicz  et al., 1986b; Dalidowicz, 1987).

         The excretion of ractopamine after oral dosing was measured in
    dogs, monkeys and pigs. A total of 79% and 70% of the administered
    single oral dose of 0.125 mg/kg bw 14C-ractopamine hydrochloride
    was recovered from the dog and monkey, respectively, during the 72 h
    collection period. In a balance-excretion study, unlabelled
    ractopamine hydrochloride administered to pigs in the feed at 20 ppm
    combined with a one-time dose of 14C-ractopamine hydrochloride of
    40 mg incorporated into control feed, was excreted almost
    quantitatively with approximately 88% in urine and 9% in faeces
    during a 7-day collection period. The bulk of the radiolabelled
    ractopamine was excreted in the first three days (95%), while 85%
    was eliminated during the first day after dosing (Williams, 1987a;
    Dalidowicz & Babbitt, 1986; Dalidowicz  et al., 1986; Dalidowicz,
    1987).

    2.1.2  Biotransformation

         A study in pigs fed 14C-ractopamine hydrochloride showed that
    the livers and kidneys of dosed animals contained three major
    metabolites of ractopamine (Metabolites A, B, and C in figure 1).
    These metabolites were formed by conjugation of the hydroxyl group
    in ring A or ring B and are chromatographically distinct (Figure 1):

    FIGURE 1


         The same three metabolites were isolated from pig urine and
    were all identified to be monoglucuronides of ractopamine. Of the
    extractable liver residues, 30-50% was found to be ractopamine and
    the rest conjugates of ractopamine. The kidneys contained a higher
    amount of conjugates (64%) and correspondingly smaller amount of
    ractopamine (20-30%). Studies in rats and dogs showed that urine
    from animals dosed with 14C-ractopamine hydrochloride contained
    the same three metabolites of ractopamine as in pigs (metabolites A,
    B, and C). These three metabolites constituted a large portion of
    14C-residues in rat and dog urine. The chromatographic profiles of
    the 14C-residue extracts of rat, dog and pig liver were
    quantitatively similar. The comparative quantitative data showing
    the mean amounts in ppm of ractopamine hydrochloride and its major
    metabolites (calculated as ractopamine hydrochloride) in kidney and
    liver tissues of pigs, dogs, and rats are summarized in Table 1
    (Dalidowicz & Babbitt, 1986; Dalidowicz, 1986a; Dalidowicz, 1986b).

        Table 1. Metabolic profile in pigs, dogs and rats
                                                                                  

                                Liver, ppm                     Kidney, ppm
                                                                                  

                           Pig     Dog     Rat             Pig     Dog     Rat
                                                                                  

    Ractopamine. HC1       0.12    0.59    0.40            0.10    0.50    0.33

    Metabolite A           0.03    0.46    0.17            0.05    0.18    0.52

    Metabolite B           0.04    0.77    0.15            0.06    0.27    0.57

    Metabolite C           0.02    1.76    0.10            0.09    0.63    0.08
                                                                                  

    Dose:     pigs (45 kg): 30 ppm in food for 4 days; pigs were killed
              12 h after the last dose;

              dogs:  0.5 mg/kg bw by gavage 3 times per day for 4 days and
              once on the fifth day; dogs were killed 6 h after the
              last dose;

              rats:  2 mg/kg bw by gavage once daily for 7 days; rats were
              killed 6 h after the last dose.
    
         The concentrations of the metabolites in dog and rat tissues
    were much higher than those in pig tissues. The nonextractable and
    the uncharacterized residues in dog and rat tissues were also much
    higher than in pig tissues (Dalidowicz, 1987).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         The results of acute toxicity studies with ractopamine are
    summarized in Table 2.

    2.2.2  Short-term studies

    2.2.2.1  Mice

         Diets containing 0, 0.02, 0.14 or 1.0% ractopamine
    hydrochloride were fed to B6C3F1 mice (10/sex/dose) for 3
    months, resulting in estimated time-weighted average daily doses of

    0, 25, 175 or 1250 mg/kg bw/day, respectively. The mice were
    observed daily for clinical signs of toxicity. Body-weights and food
    consumption were measured weekly in all animals. Necropsies were
    performed on all mice sacrificed at the end of the study. A set of
    tissues was examined microscopically.

        Table 2. Acute toxicity studies
                                                                                  

    Species      Sex    Route               LD50 (mg/kg/bw       Reference
                                                                                  

    Mouse        M      oral                3547 (2912-4321)     Williams et al.,
                                                                 1985a
                 F      oral                2545 (2219-2919)     Williams et al.,
                                                                 1985a

    Rat          M      oral                474 (398-564)        Williams et al.,
                                                                 1985b
                 F      oral                367                  Williams et al.,
                                                                 (1985b)

    Rat          M      intraperitoneal     132 (110-159)        Williams et al.,
                 F      intraperitoneal     122 (102-145)        1985c

    Rabbit       M      dermal              >2000                Williams et al.,
                 F                                               1984a

    Rat          M      inhalation          LC50 (4 h): 2.8      Williams et al.,
                 F                          mg/L                 1985k
                                                                                  
    

         All of the treated animals survived the treatment period.
    Alopaecia, a common finding in B61C3F1 mice, was observed in
    22 animals of each sex. In this study the occurrence of alopaecia
    was markedly decreased in the 1250 mg/kg bw/day group compared to
    the other groups. No adverse signs of toxicity were attributed to
    treatment with ractopamine hydrochloride. Male mice in the 25 and
    175 mg/kg bw/day groups had a significant increase in cumulative
    weight gain in the last eight weeks of the study. Differences in the
    final mean body-weights of the treatment groups compared to the
    controls were not statistically significant.

         Treatment- and dose-related mild to moderate increases occurred
    in erythrocyte counts, haemoglobin concentrations, and packed cell
    volumes in both sexes of the 175 and 1250 mg/kg bw/day groups. Minor
    changes occurred in the mean corpuscular volume and mean corpuscular
    haemoglobin concentration in males of the 1250 mg/kg bw/day group.
    The changes in thrombocyte counts were minimal in both sexes of the
    1250 mg/kg bw/day group. All other haematologic parameters were
    similar to control values. There were no treatment-related effects
    on any haematologic parameters in mice of the 25 mg/kg bw/day group.

         Treatment-related clinical chemistry changes consisted of mild
    increases in urea nitrogen and cholesterol concentrations in males
    of the 1250 mg/kg bw/day group. Female mice in both the 175 and 1250
    mg/kg bw/day groups had mild increases in urea nitrogen and
    cholesterol concentrations. The decrease in serum sodium
    concentration in female mice of the 1250 mg/kg bw/day group,
    although statistically significant, was minimal and the value fell
    within the normal range. No alteration in the other electrocytes
    concentration occurred. All other clinical chemistry parameters were
    similar to control values. There were no treatment-related effects
    on serum chemistry in mice of the 25 mg/kg bw/day group.

         In males, a decrease in testicular weights, both absolute and
    relative, occurred in a dose-dependent manner. Both males and
    females in the 1250 mg/kg bw/day group had increased absolute and
    relative heart weights. All the other organ weights were similar to
    control values.

         Microscopically, treatment-related changes involved the
    periaortic and intercapsular brown fat in mice of both sexes in the
    1250 mg/kg bw/day group. The plasma membranes of the affected brown
    fat cells from the 1250 mg/kg bw/day group of mice were more
    discrete and stained intensely with eosin. The cytoplasm of these
    cells was filled with uniformly small spherical vacuoles, the
    margins of which also stained intensely with eosin. These changes
    were diagnosed as marked cytoplasmic changes. The cytoplasm of the
    brown fat cells with minimal change was characterized by small
    vacuoles, but the margins of the vacuoles did not stain intensely
    with eosin. Brown fat in female mice and a few male mice of the
    control and of the 25 and 175 mg/kg bw/day groups was characterized
    as having a minimal cytoplasmic change. Due to the dose-dependent
    decrease in testicular weights, a NOEL could not be established in
    this study (Williams  et al., 1985d).

    2.2.2.2  Rats

         Diets containing 0, 20, 200 or 2000 ppm ractopamine
    hydochloride were fed to Fischer 344 rats (20/sex/dose) for three
    months. The time-weighted average doses were 0, 1.3, 13, or 153
    mg/kg bw/day for males and 0, 1.4, 15, or 157 mg/kg bw/day for

    females. The rats were observed daily for clinical signs of
    toxicity. Body-weights and food consumption were measured weekly for
    all animals. Autopsies were done on animals at the end of the study.
    Histological examination of tissue sections was conducted on all
    tissues.

         All of the treated animals survived the treatment period. The
    clinical signs observed were decreased body-weight gain, increased
    food consumption and decreased efficiency of food utilization. These
    signs were observed in the 155 mg/kg bw/day group only, where they
    were observed throughout the study in rats of both sexes. In
    addition to these signs decreased serum triglyceride and cholesterol
    levels and increased serum urea nitrogen concentrations were
    observed.

         An increase in serum potassium was observed in the 155 mg/kg
    bw/day group in both sexes. Total erythrocyte count, haemoglobin
    concentration, and packed cell volume all increased in rats of the
    155 mg/kg bw/day group, as expected with a beta-agonist. There was a
    decrease in uterine weight in rats of the 155 mg/kg bw/day group in
    this study and a slight reduction in spleen weight in the 13-15 and
    155 mg/kg bw/day groups. No change was observed in heart weight of
    any treated animals and there was no microscopic evidence of
    myocardial necrosis, which is a well-documented effect following
    large doses of epinephrine and other sympathomimetic amines.

         Histopathology examination of brown fat revealed slight to
    moderate cytoplasmic change in the 13-15 and 155 mg/kg bw/day
    groups. The NOEL for males was 1.3 mg/kg bw/day and 1.4 mg/kg bw/day
    for females, based on the absence of biochemical or histological
    changes (Williams  et al., 1985e).

    2.2.2.3  Dogs

         Groups of 4 male and 4 female beagle dogs were given total
    doses of 0.112, 0.224 or 5.68 mg/kg bw/day of ractopamine
    hydrochloride in gelatin capsules for 1 year. The doses were given
    in capsules as three equal divided doses every 6 h. This study was
    based on pilot studies in dogs, in which an oral dose of 0.05 mg/kg
    bw/day was associated with skin and oral mucosal reddening and a
    dose of 0.035 mg/kg bw/day was the NOEL. On the basis of this NOEL,
    the frequency of dosing was increased to three times per day, 6 h
    apart, in order to triple the daily exposure to 0.112 mg/kg bw/day
    for the low-dose. The middle-dose, 0.224 mg/kg bw/day (0.075 mg/kg
    bw, 3 times per day), was selected as a minimal cardiostimulatory
    dose. The high-dose, 5.68 mg/kg bw/day (1.89 mg/kg bw, 3 times per
    day) was selected because it produced near maximal cardiostimulation
    at 2 h following a single oral dose of 1.5 mg/kg bw, in the pilot
    study.

         All dogs survived the one-year treatment period. Body-weight,
    food consumption, ophthalmic examination, electrocardiogram wave
    forms, bone marrow evaluation and urinalysis results were not
    affected by treatment. The zero-time resting heart rate means for
    the study (collected on test-days 29, 68, 83, 119, 155, 182, 211,
    273 and 366) for low-, middle- and high-dose dogs (sex combined)
    were 123, 116 and 115 beats per minute, respectively, and all were
    significantly (p < 0.01) depressed compared to 141 beats per minute
    for the controls.

         Clinical signs of treatment-related effects were transient
    cutaneous erythema and oily and unkept hair coat in the high-dose
    dogs. Cutaneous erythema occurred in the mid-dose group in the first
    five months of the study. Erythrocyte number, haemoglobin
    concentration and packed cell volume were significantly decreased in
    the high-dose group starting at 90 days, which and continuing up to
    the end of the study. Several clinical chemistry parameters were
    altered in the high-dose group by 90 days, which persisted until the
    termination of the study. Serum potassium and urea nitrogen
    concentration increased while serum glucose, cholesterol and
    triglyceride concentration decreased.

         Treatment-related gross and histopathological findings were
    limited to the high-dose group. Both absolute and relative heart
    weight and absolute thyroid and adrenal weights were significantly
    decreased. Grossly, there was a slight decrease in abdominal and
    thoracic fat. Microscopically, a decrease in hepatic centrilobular
    glycogen and an increase in perithymic and periaortic brown fat was
    noted in the high-dose dogs.

         The appearance of bradycardia at all doses, most prominent
    during the first 6 months, precluded a NOEL being established in
    this study (Williams, 1987b).

    2.2.2.4  Monkeys

         Rhesus monkeys (2/sex/dose) were given ractopamine
    hydrochloride dissolved in nanopure water at doses of 0.25, 0.5 or
    4 mg/kg bw once daily by nasogastric gavage in a volume of 1 ml/kg
    bw for 6 weeks. Control monkeys received vehicle (nanopure water)
    only. This 6-week subchronic study was conducted in rhesus monkeys
    to determine the doses at which toxic effects occurred so that
    appropriate effect and no-effect doses can be selected for a 1-year
    study in monkeys.

         Treatment had no significant toxicological effect on
    body-weight, food consumption, ophthalmic examination, daily
    clinical examination, electro-cardiogram wave forms, haematology,
    clinical chemistry, or urinalysis parameters. No treatment-related
    abnormalities were observed at the final physical examination. There

    was no induction of the hepatic enzyme p-nitroanisole o-demethylase,
    and no compound-related gross lesions were observed at necropsy. No
    organ weight changes were observed except for increased salivary
    gland weight relative to body-weight (sexes combined) in the 4 mg/kg
    bw/day group. The microscopic appearance of salivary gland and heart
    from monkeys of the 4 mg/kg bw/day group was similar to that of
    controls. Microscopic evaluation of the remaining tissues collected
    by necropsy and determination of heart and lung -adrenergic
    receptor numbers was in progress at the time of the review.

         Monkeys given 4 mg/kg bw/day ractopamine hydrochloride
    developed daily tachycardia, which was maximal by 0.5 h post-dose,
    and remained elevated through 16 h after dosing. The monkeys did not
    demonstrate the significant slowing of the night-time heart rates as
    seen in the control, 0.25 and 0.5 mg/kg bw/day groups of monkeys. A
    slight tachyphylaxis to ractopamine-induced heart rate stimulation
    was apparent after the first day, which did not progress with time
    of treatment. No effect on the electrocardiogram wave forms or
    cardiac histopathology occurred in the presence of marked daily
    tachycardia during the 6 weeks at 4 mg/kg bw/day. The NOEL was
    0.5 mg/kg bw/day (Williams  et al., 1985h).

         Two groups, each composed of three male and three female rhesus
    monkeys, were administered either vehicle or 0.125 m-g ractopamine
    hydrochloride/kg bw once per day for 90 days by nasogastric gavage.
    The test substance was prepared as a solution in nanopure water at a
    concentration of 0.125 mg/ml.

         One ml/kg bw of vehicle or the ractopamine solution was
    administered and the monkeys were maintained in a conscious state
    during the dosing and the cardiovascular monitoring procedures.

         All of the monkeys were clinically normal throughout the study.
    There were no effects on body-weight, food consumption, heart rate,
    or on the electro-cardiographic wave forms in any control or treated
    monkeys. The NOEL was determined to be 0.125 mg/kg bw/day
    (Williams  et al., 1985i). .

         Rhesus monkeys, 2/sex/group, were exposed to mean aerosol
    concentrations of 0 (air, control), 0.05, 0.17, or 0.44 mg
    ractopamine hydrochloride/m3 of air for 4 h per day for 8 days
    over a 10-day period (excluding week-ends). The activity median
    equivalent aerodynamic diameters (AMEAD) of the aerosols were 5.6,
    6.2 and 6.4 m for groups exposed to 0.05, 0.17 or 0.44 mg
    ractopamine hydrochloride/m3. There was a slight increase in
    post-exposure (night-time) heart rates. No treatment-related changes
    occurred in body-weights, organ weights, food consumption,
    haematology, or clinical chemistry parameters. There were no
    treatment-related gross or microscopic lesions. The
    no-observed-effect exposure concentration was 0.17 mg ractopamine
    hydrochloride/m3 (Williams  et al., 1985f).

         Rhesus monkeys, 2/sex/group, were exposed to mean aerosol
    concentrations of 0.38, 1.69, 6.42 or 23.8 mg ractopamine
    hydrochloride/m3 for 4 h per day for 2 to 8 days over an 18-day
    period (excluding weekends) following a 7-day period of control
    heart rate data collection. The AMEAD of the aerosols were 6.2, 7.8,
    8.3 and 11.4 m for the four exposure groups, respectively.
    Treatment was stopped after 2 exposures at 23.8 mg ractopamine
    hydrochloride/m3 and after 7 exposures at 1.69 and 6.42 mg
    ractopamine hydrochloride/m3. The 0.38 mg ractopamine
    hydrochloride/m3 exposure group received no exposure until the
    last 8 days of the study prior to necropsy. Two animals were
    sacrificed prior to study termination due to rejection of the
    implanted ECG transmitter. All other animals survived to termination
    of the study. No clinical signs of toxicity were observed. Increased
    heart rates were observed in all exposure groups during exposure
    (daytime) and following exposure (night-time). The increased daytime
    and night-time heart rates persisted after treatment was stopped and
    required approximately two weeks to return to normal values. No
    treatment-related changes occurred in body-weights, organ weights,
    food consumption, haematology or clinical chemistry parameters. No
    treatment-related gross or microscopic lesions were observed. A
    no-observed-effect exposure concentration for inhalation of
    ractopamine hydrochloride aerosol could not be determined in this
    study (Williams  et al., 1985g).

    2.2.3  Long-term/carcinogenicity studies

              No infomation available.

    2.2.4  Reproduction studies

    2.2.4.1  Rats

         Groups of 25 male and 25 female Crl:CD(SD)BR rats, about 35
    days old at the beginning of the study were maintained on diets
    containing 0, 2, 20, 200 or 2000 ppm ractopamine in two generations
    of parental rats. Weanling F0 male rats were maintained on the
    test diets for 70 days prior to mating and the female rats were on
    the test diets for the 14 days prior to mating.

         The F0 parental rats were mated once and the females were
    allowed to deliver and rear their young through weaning. However,
    F1 animals were mated for two breeding trials. In the first
    breeding trial, the females were allowed to deliver and rear their
    progeny through postpartum day 21. In the second breeding trial, the
    females were killed on gestation day 20 for the assessment of
    intrauterine reproduction parameters and the collection of fetuses
    for external, visceral and skeletal examination. Significance was
    defined as P <0.05 in this study.

         Significant treatment-related depression in body-weight and
    body-weight gain occurred in both F0 and F1 males of the 2000
    ppm group. Significant depression in body-weight also occurred in
    F1 females of the 2000 ppm group. Food consumption was
    significantly depressed in F1 males of the 2000 ppm group only.

         Mating performance and fertility were comparable with the
    controls at each treatment level in each litter of the F0 (F1a)
    and F1 (F2a and F2b) generations.

         During the breeding trials for F1a and F2a litters,
    gestation length was not affected; however, the mean litter size and
    mean progeny survival indices were depressed significantly in the
    2000 ppm group.

         Significant reductions in the mean weanling weight of F1a and
    F2a pups born to dams treated at 2000 ppm from days 1 through 21
    postpartum were recorded. The percentage of male progeny was
    significantly depressed only in the F1a litters.

         Clinical signs such as pallor, apparent hypothermia, thinness,
    dehydration and rough hair coat occurred with the highest frequency
    in the neonatal and postnatal progeny of the 2000 ppm group.
    Seventy-three pups of the F1a generation from the 2000 ppm group
    were either born dead or died during the lactation period. Gross
    external and internal examinations of the pups revealed that 13 of
    these pups had abnormalities which included oedema (8), cleft
    palate (7), limb abnormalities (5), brachygnathia (3), protruding
    tongue (3), open eyelids (3), omphalocele (2), clotted blood in the
    lateral ventricles of the brain (1), microphthalmia (2), and
    enlarged heart (1).

         Malformations were observed in 0, 1, 0, 2 and 63 F2a fetuses
    from dams treated at 0, 2, 20, 200 and 2000 ppm, respectively.
    However, significant increases in malformations were recorded in the
    63 pups from the dams treated at 2000 ppm. These abnormalities
    included oedema, hydraminos, cleft palate, protruding tongue, short
    limbs, missing digits, open eyelids, brachygnathia, fused digits,
    limb abnormalities (wavy humerus radius, ulna, femur, tibia and
    fibula, mishapen scapula, micromelia, kinked tail, enlarged heart,
    kyphosis adactyly (hindlimb), and omphalocele.

         Percent early, late and total resorption were significantly
    increased in dams treated at 2000 ppm. In dams of the 200 ppm and
    2000 ppm groups, significant depression in the proportion of normal
    fetuses was observed. In the 2000 ppm group, this depression was
    accompanied by statistically-significant proportions of fetuses with
    developmental variations and abnormalities.

         In view of the fact that significant teratological effects were
    observed only at the highest dose level tested, a dose level at
    which maternal toxicity was also observed, it was concluded that the
    NOEL was 200 ppm, equal to 15 mg/kg bw/day (Williams & Hoyt, 1986).

    2.2.5  Special studies on genotoxicity

         The results of a limited series of  in vitro and  in vivo
    genotoxicity studies on ractopamine are summarized in Table 3. All
    results were reported to be negative.

    2.3  Observations in humans

         A pilot clinical trial evaluating bronchodilator activity of
    ractopamine in oral dosage form and as aerosol was conducted in
    1960. Ractopamine was administered orally to four patients with
    chronic bronchial asthma at doses of 30 and 45 mg. No clear-cut
    evidence of bronchodilator activity, CNS stimulation, or increase in
    pulse rate was seen in these patients. Two patients receiving 30 to
    45 mg of ractopamine showed 15 to 20 mm Hg systolic elevation of
    blood pressure lasting for about 1 h. A summary on the use of
    50 mg/ml ractopamine with a DeVilbiss nebulizer in four asthmatic
    patients indicated that on a 0 to 4 scale of effectiveness according
    to patients, ractopamine scored 3, 3, 1 and 0 in these patients. It
    was stated that ractopamine as an aerosol has failed to show any
    activity at doses up to 6 mg (Shipley, 1960).

        Table 3: Results of genotoxicity assays on ractopamine
                                                                                               

                                              Concentration of
    Test System           Test Object         Ractopamine         Results    References
                                                                                               

    Unscheduled           Fischer 344 rat     0.5-1000 g/ml      Negative   Williams et al.,
    DNA synthesis         hepatocytes                                        1984b
    in vitro

    Ames test (1)         S. typhimurium      50-5000 g/ml       Negative   Williams et al.,
                          TA1535, TA1537,                                    1984c
                          TA1538, TA98,
                          TA100

    Ames test (1)         S. typhimurium      0.1-1000 g/ml      Negative   Williams &
                          G46, TA1535,                                       Thompson,
                          TA100, C3076,                                      1984
                          TA1537, D3052,
                          TA1338, TA98
                          E. coli
                          WP2, WP2uvrA-

    Mouse lymphoma cell   L5178Y mouse        10-350 g/ml        Negative   Williams et al.,
    thymidine kinase                                                         1984d

                                              100-700 g/ml

    Sister chromatid      Chinese hamsters    200-500 mg/kg       Negative   Williams et al.,
    exchange in bone                                                         1985j
    marrow of hamsters
    in vivo
                                                                                               

    (1)  With and without rat liver S-9 fraction.
    
    3.  COMMENTS

         Results of various studies were reviewed by the Committee,
    including pharmacokinetic, biotransformation, acute and short-term
    toxicity, reproductive, teratogenicity, and genotoxicity studies and
    a limited number of studies in humans.

         Studies with 14C-ractopamine in several species have
    indicated rapid absorption following oral administration. In pigs,
    labelled ractopamine was excreted almost quantitatively;
    approximately 88% was recovered in urine and 9% in faeces during a
    7-day period. Studies in pigs, rats, and dogs fed 14C-ractopamine
    showed three major metabolites, identified as monoglucuronides of
    ractopamine.

         In acute studies, ractopamine was substantially more toxic
    orally to the rat (LD50 approximately 450 mg/kg bw) than the mouse
    (LD50 approximately 3000 mg/kg bw). 

         The genotoxic potential of ractopamine was evaluated in a
    limited series of  in vitro and  in vivo studies, all of which
    were reported to be negative. However, in the absence of a
    carcinogenicity study, and as human exposure is likely to be
    extensive, the Committee concluded that additional genotoxicity
    testing would be desirable.

         The short-term toxicity of ractopamine has been evaluated in
    mice, rats, dogs, and monkeys. Ractopamine was fed to B6C3F1
    mice for 3 months at doses of 25, 175, or 1250 mg/kg bw/day. The
    most significant effect noted was a dose-dependent decrease, both
    absolute and relative, in testicular weights. In both males and
    females in the highest-dose group, absolute and relative heart
    weights were increased. However, no histopathological changes were
    observed in either the heart or the testes. A clear NOEL could not
    be established in this study.

         Fischer 344 rats were fed doses up to approximately 155 mg/kg
    bw/day for 3 months. The highest-dose group showed decreased
    body-weight gain, increased food consumption, decreased efficiency
    of food utilization, and an increase in serum potassium
    concentration. There was a decrease in uterine weight in rats of
    this group and a slight reduction in spleen weight in the top two
    groups. The NOEL was 1.3 mg/kg bw/day in this study.

         Beagle dogs were given three doses daily, 6 h apart, totalling
    0.112, 0.224 or 5.68 mg/kg bw/day of ractopamine in gelatin capsules
    for 1 year. Treatment-related minor histopathological findings were
    limited to the high-dose group and to the liver. The occurrence of
    mild nocturnal bradycardia, most prominent during the first

    6 months, meant that a clear no-effect level was not observed in
    this study. The Committee noted that the quantity of ractopamine
    residues consumed in 500 g of meat from animals slaughtered without
    a withdrawal period would closely approach an ADI derived from the
    lowest dose of 0.112 mg/kg bw/day and a safety factor of 100.

         In a further study of cardiovascular effects, rhesus monkeys
    were given either vehicle or ractopamine at 0.125 mg/kg bw/day for
    90 days by gavage. This dose was 2.5 times the single dose known to
    produce tachycardia and peripheral vasodilation in the dog. In
    addition, the selected dose exceeded the total daily dose
    (0.112 mg/kg bw/day) associated with nocturnal bradycardia in the
    1-year dog study. The NOEL for this study was 0.125 mg/kg bw/day.

         Rhesus monkeys were given ractopamine at doses of 0.25, 0.5, or
    4 mg/kg bw/day once daily by gavage for 6 weeks to determine the
    doses to be used in a 1-year study. Monkeys given 4 mg/kg bw/day
    developed daily tachycardia which was maximal by 30 min agter
    dosing, and continued for 16 h. Monkeys in the group did not
    demonstrate the significant slowing of the nocturnal heart rates
    seen in the other groups. The NOEL for this study was 0.5 mg/kg
    bw/day.

         The effect of ractopamine on reproductive and developmental
    performance in Sprague-Dawley rats was evaluated at dosage levels of
    up to 2000 ppm in the diet. Significant effects, which included a
    reduction in mean litter size and an increase in the total number of
    resorptions, were restricted to the high dose which was also
    maternally toxic. The NOEL was 200 ppm, equal to 15 mg/kg bw/day, in
    this study. The Committee considered the teratogenicity segment of
    this study to be adequate to assess developmental toxicity. A minor
    teratogenic response was observed only at the highest dose
    (2000 ppm) tested, at which maternal toxicity was also noted.

         The bronchodilator and inotropic effects of ractopamine were
    evaluated in pilot clinical trials in humans. Four patients showed
    little evidence of bronchodilator activity, central nervous system
    stimulation, or an increase in pulse rate. Two patients showed a
    mild elevation of blood pressure lasting for about 1 hour. An
    infusion study showed inotropic and chronotropic enhancement in both
    healthy volunteers and heart patients.

    4.  EVALUATION

         The Committee concluded that, on the basis of the short-term
    studies available, residues of ractopamine appeared to have little
    toxic potential for the consumer. The effects recorded were in the
    main those to be expected from a -adrenoceptor agonist. It might
    therefore be appropriate to assess ractopamine on the basis of a
    NOEL for pharmacological effects that are relevant to its ingestion
    by humans as a residue in edible meats. However, because such a NOEL
    could not be determined in the 1-year study in dogs, the Committee
    was unable to establish an ADI.

         The Committee noted that: (a) some -adrenoceptor agonists were
    carcinogenic; (b) no long-term studies had been conducted in
    rodents; and (c) there were no data relating to the long-term
    exposure of humans to ractopamine. Therefore, before reviewing the
    compound again, the Committee would wish to see evidence and
    arguments in at least the following areas:

    1.   Genotoxicity

         -    A further  in vivo study such as a micronucleus test.

    2.   Pharmacology

         -    investigations that fully explore the pharmacological
              properties of ractopamine;

         -    the relative contributions of 1- and 2-adrenoceptor
              activation to the spectrum of effects produced by
              ractopamine;

         -    a sufficient basis from which to establish the most
              sensitive indicator (test and species) of the
              pharmacological effects of ractopamine;

         -    validation of the utility of this indicator in the setting
              of a pharmacological NOEL for humans;

         -    a survey of the pharmacokinetic parameters of
              -adrenoceptor agonists in humans and laboratory species,
              including those relevant to oral administration;

         -    determination of appropriate timing for observations in
              animal studies to reveal both the onset and the peak
              values of all relevant effects.

    3.   Human data

         -    A survey of all non-therapeutic effects that follow
              long-term -adrenoceptor agonist use in humans, to assist
              in the prediction of the consequences of the long-term
              intake of residues of ractopamine by consumers of animal
              meat.

         Depending on the results of the above investigations, it may be
    necessary to perform other studies to explore further the potential
    carcinogenicity of ractopamine.

    5.  REFERENCES

    DALIDOWICZ, J.D. (1986a). Metabolism of 14C-ractopamine HCl in the
    rat. Unpublished Report No. ABC-0285 from Lilly Research
    Laboratories, Division of Eli Lilly and Company, Greenfield,
    Indiana, USA. Submitted to WHO by Elanco Products Company, Division
    of Eli Lilly and Company, Indianapolis, IN, USA.

    DALIDOWICZ, J.D. (1986b). Metabolism of 14C-ractopamine HCl in the
    dog. Unpublished Report No. ABC-0301 from Lilly Research
    Laboratories, Division of Eli Lilly and Company, Greenfield,
    Indiana, USA. Submitted to WHO by Elanco Products Company, Division
    of Eli Lilly and Company, Indianapolis, IN, USA.

    DALIDOWICZ, J.D. & BABBITT, G.E. (1986). Characterization of
    14C residues in tissues and excreta from swine fed
    14C-ractopamine HCl. Unpublished Report No. ABC-0355 from Lilly
    Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    DALIDOWICZ, J.E., THOMSON, T.D. & HERBERG, R.J. (1986).
    14C-ractopamine HCl balance - excretion study in swine.
    Unpublished Report No. ABC-0330 from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Greenfield, Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly
    and Company, Indianapolis, IN, USA.

    DALIDOWICZ, J.D. (1987). Comparative metabolism of 14C-ractopamine
    HCl in swine, dogs, and rats. Unpublished Report No. ABC-0369 from
    Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    SHIPLEY, R.E. (1960). Summary for research project committee May 31,
    1960. Unpublished memoranda from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Greenfield, Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly
    and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D. & THOMPSON, C.Z. (1984). The effect of compound
    31537, EL-737 on the induction of bacterial mutation using a
    modification of the Ames test. Unpublished Report No. 840507GPA2000
    from Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., NEGILSKI, D.S. & MARKEY, T.F. (1984a). The acute
    dermal, ocular, and inhalation toxicity of compound 31537 (EL-737).
    Unpublished Reports Nos. B-D-82-84, B-E-108-84 and R-H-47-84 from
    Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., HILL, L.E. & PROBST, G.S. (1984b). The effect of
    compound 31537, EL-737, on the induction of DNA repair synthesis in
    primary cultures of adult rat hepatocytes. Unpublished Reports Nos.
    840503UDS2000 and 840508UDS2000 from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Greenfield, Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly
    and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., REXROAT, M.A. & PROBST, G.S. (1984c). The effect of
    EL-737 (compound 31537) the induction of reverse mutations in
     Salmonella typhimurium using the Ames test. Unpublished Report No.
    840716AMS2000 from Lilly Research Laboratories, Division of Eli
    Lilly and Company, Greenfield, Indiana, USA. Submitted to WHO by
    Elanco Products Company, Division of Eli Lilly and Company,
    Indianapolis, IN, USA.

    WILLIAMS, G.D., OBERLY, T.J., BEWSEY, B.J. & PROBST, G.S. (1984d).
    The effect of 31537 (EL-737) on the induction of forward mutation at
    the thymidine kinase locus of L517Y mouse lymphoma cells.
    Unpublished Report No. 840627MLA2000 from Lilly Research
    Laboratories, Division of Eli Lilly and Company, Greenfield,
    Indiana, USA. Submitted to WHO by Elanco Products Company, Division
    of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R., BRIDGE, T.L. (1985a). The acute
    toxicity of compound 31537 (EL-737) administered orally to the ICR
    mouse. Unpublished Reports Nos. M-0-178-84 and M-0-179-84 from Lilly
    Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985b).  The acute
    toxicity of compound 31537 (EL-737) administered orally to the
    Fischer 344 rat. Unpublished Report No. R-0-132-84 and R-0-133-84
    from Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985c). The acute
    toxicity of compound 31537 (EL-737) administered intraperitoneally
    to the Fischer 344 rat. Unpublished Reports Nos. R-P-08-84 and
    R-P-09-84 from Lilly Research Laboratories, Division of Eli Lilly
    and Company, Greenfield, Indiana, USA. Submitted to WHO by Elanco
    Products Company, Division of Eli Lilly and Company, Indianapolis,
    IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985d). A three-month
    toxicity study of ractopamine hydrochloride fed in the diet to
    B6C3F1 mice. Unpublished Report No. M01584 from Lilly Research
    Laboratories, Division of Eli Lilly and Company Greenfield, Indiana,
    USA. Submitted to WHO by Elanco Products Company, Division of Eli
    Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985e). A three-month
    toxicity study of 031537 (EL-737) administered orally to Fischer 344
    rats. Unpublished Report No. R06184 from Lilly Research
    Laboratories, Division of Eli Lilly and Company, Greenfield, Indiana
    USA. Submitted to WHO by Elanco Products Company, Division of Eli
    Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985f). 8-Day
    inhalation study in rhesus monkey. Unpublished Report No. P06288
    from Lilly Research Laboratories, Division of Eli Lilly and Company,
    Lilly Corporate Center, Indianapolis, Indiana, USA. Submitted to WHO
    by Elanco Products Company, Division of Eli Lilly and Company,
    Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R. & BRIDGE, T.L. (1985g). 18-Day
    inhalation study in rhesus monkey. Unpublished Report No. P00388
    from Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R., & BRIDGE, T.L. (1985h). An interim
    summary of a 6-week subchronic toxicity study of ractopamine
    hydrochloride administered by nasogastric gavage to rhesus monkeys.
    Unpublished Report No. P00691 from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Lilly Greenfield, Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly,
    and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., McKINLEY, E.R., & BRIDGE, T.L. (1985i). The effect
    of subchronic administration of ractopamine hydrochloride on heart
    rate and electrocardiographic waveforms in conscious rhesus monkeys.
    Unpublished Report No. P02186 from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Greenfield Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly
    and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., NEAL, S.B. & PROBST, G.S. (1985j). The effect of
    compound 31537 (EL-737) on the  in vivo induction of sister
    chromatid exchange in bone marrow of Chinese hamsters. Unpublished
    Report No. 850121SCE2000 from Lilly Research Laboratories, Division
    of Eli Lilly and Company, Greenfield, Indiana, USA. Submitted to WHO
    by Elanco Products Company, Division of Eli Lilly and Company,
    Indianapolis, IN, USA.

    WILLIAMS, G.D., NEGILSKI, D.S. & BRIDGE, T.L., MARKEY, T.F. (1985k).
    The acute oral, dermal, ocular and inhalation toxicity of a granular
    premix formulation (AFN-026) containing 10% compound 31537 (EL-737).
    Unpublished Reports Nos. R-0-188-85, B-D-104-85, B-E-120-85, and
    R-H-066-85 from Lilly Research Laboratories, Division of Eli Lilly
    and Company, Greenfield, Indiana, USA. Submitted to WHO by Elanco
    Products Company, Division of Eli Lilly and Company, Indianapolis,
    IN, USA.

    WILLIAMS, G.D., HOYT, J.A. (1986). An eleven-month two-generation
    reproduction study, including a teratology segment, in Cd rats
    maintained on diets containing ractopoamine hydrocholoride (EL-739,
    Compound 31537). Unpublished report for study Nos. R11385 and R18985
    from Lilly Research Laboratories, Division of Eli Lilly and Company,
    Greenfield, Indiana, USA. Submitted to WHO by Elanco Products
    Company, Division of Eli Lilly and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D., (1987a). Comparative bioavailability of
    14C-ractopamine hydrochloride (031537, EL-737) following a single
    oral dose of 0.125 mg/kg in the dog and monkey. Unpublished Reports
    Nos. D04686 and P03086 from Lilly Research Laboratories, Division of
    Eli Lilly and Company, Greenfield, Indiana, USA. Submitted to WHO by
    Elanco Products Company, Division of Eli Lilly and Company,
    Indianapolis, IN, USA.

    WILLIAMS, G.D., (1987b). A chronic toxicity study of ractopamine
    hydrochloride administered orally to beagle dogs for one year.
    Unpublished Report No. D05885 from Lilly Research Laboratories,
    Division of Eli Lilly and Company, Greenfield, Indiana, USA.
    Submitted to WHO by Elanco Products Company, Division of Eli Lilly
    and Company, Indianapolis, IN, USA.

    WILLIAMS, G.D. & POHLAND, R.C. & BYRD, T.K. (1987). Bioavailability
    of radiocarbon following the administration of single oral doses of
    14C-EL-737 (31537) to F344/N Hsd BR rats. Unpublished Reports Nos.
    R03985, RO4085 and RO4185 from Lilly Research Laboratories, Division
    of Eli Lilly and Company, Greenfield, Indiana, USA. Submitted to WHO
    by Elanco Products Company, Division of Eli Lilly and Company,
    Indianapolis, IN, USA.


    See Also:
       Toxicological Abbreviations
       Ractopamine (WHO Food Additives Series 53)
       RACTOPAMINE (JECFA Evaluation)