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    ZERANOL

    EXPLANATION

         Zeranol was considered at the twenty-sixth meeting of the Joint
    FAO/WHO Expert Committee on Food Additives (Annex 1, reference 59),
    but it could not be evaluated at that time because the necessary
    documentation on residue levels, good animal husbandry in relation to
    the use of the agent, and details of methods of analysis were not
    available. At the twenty-seventh meeting (Annex 1, reference 62), the
    Committee provisionally accepted the use of zeranol as an anabolic
    agent for the production of meat for human consumption in accordance
    with good animal husbandry practice, and requested submission of the
    results of (1) a study known to be in progress to establish a
    no-effect level for hormonal activity in non-human primates and (2)
    adequate carcinogenicity studies in two rodent species.

         This monograph contains the data previously considered by the
    Committee, as well as data that have been submitted recently.

    BIOLOGICAL DATA

    Toxicological studies

    Special studies on carcinogenicity

    Mice

         Four groups of 50 male and 50 female CDI mice each were
    maintained on diets containing 0, 0.15, 1.5, or 15 ppm zeranol for 104
    weeks. Another group of 50 male and 50 female CDI mice, which served
    as a positive control, were maintained on a test diet containing
    2.5 ppm estradiol-17. Haematology studies on 10 males and 10
    females/group were carried out at weeks 26, 52, and 78 and on all
    surviving animals at week 104. At week 104 of the study all surviving
    mice were killed and autopsied. Full histopathological examinations
    were performed on all control, highest-dose zeranol, and estradiol-17
    test animals, as well as on all animals dying during the course of the
    study. Partial histopathological examinations (adrenals, kidneys,
    liver, lungs, mammary gland, uterus, vagina, cervix, prostate, seminal
    vesicles, testes, pituitary, skin, and all masses seen directly) were
    performed on all other animals.

         Mortality in the animals treated with zeranol was slightly higher
    than in controls. However, mortality was markedly increased in the
    animals fed estradiol-17, particularly in females. Male mice treated
    with zeranol showed a slight decrease in body weight compared to
    control mice during the first 52 weeks of the study, but the effect
    was not apparent during the second 52-week period. In contrast, males
    treated with estradiol-17 showed a significant decrease in body
    weight throughout the course of the study. Females on the test diets
    showed similar weight gains to the controls. The levels of food
    consumption of male and female mice on diets containing zeranol and of
    males receiving estradiol-17 were similar to controls. Females
    receiving estradiol-17 showed increased food consumption (from week
    80) compared to the control animals.

         During the course of the study, there were minor changes in
    haematological parameters in the mice treated with zeranol. Dosing
    with estradiol-17 resulted in a reduction in haemoglobin levels and
    red blood cell count in males, and changes in the differential white
    blood cell patterns in females. At autopsy, a few significant changes
    were observed in the mice fed 15 ppm zeranol, namely pituitary
    enlargement and dilation of the seminal vesicles in males. In female
    mice there was an increased incidence of alopecia. The incidences of
    these changes were greatly increased in mice in the positive control
    group. Histopathological examination of the highest-dose zeranol-
    treated mice showed brown degeneration in the adrenals in both sexes
    and conversion of the submaxillary salivary glands to the female type,

    trabeculation in the sterum, and dilation of the seminal vesicles in
    males. These estrogenic effects were very mild compared to those in
    mice fed estradiol-17. In the 0.15 ppm, but not the 1.5 ppm, group
    the incidences of ovaries with corpora lutea present and no secondary
    follicles (females) and dilatation of the seminal vesicles (males)
    were significantly decreased compared with controls. However, these
    effects were not considered to be true hormonal effects, because (1)
    the effect on the ovaries was not dose related and (2) the lower
    incidence of dilatation of the seminal vesicles was more common in
    older animals, and the lower incidence in treated animals probably was
    related to the longer average survival time of the controls. Other
    significant effects observed in the 15 ppm zeranol group included an
    increase in mucous cervical and vaginal epithelium (20/46 compared to
    10/48 in controls; 0/47 in the positive control group) and subcapsular
    hyperplasia of the adrenals in males (30/49 compared to 16/50 in
    controls). The only tumourogenic effect of zeranol noted in this study
    was in the pituitary gland of males in the highest-dose group. The
    incidences of anterior lobe adenomas and hyperplasia plus tumours in
    the anterior lobe noted in this study are summarized in Table 1.

    Table 1.  Incidences of hyperplasia and rumours of the pituitary gland
              in male mice
                                                                        

                       Negative   Zeranol (ppm in the diet)     Positive
    Finding            control    0.15      1.5       15        control
                                                                        

    Anterior lobe      1          0         0         8         28
    adenoma

    Hyperplasia +      1          2         2         12        33
    rumours in
    anterior lobe
                                                                        

         These tumours rarely occur spontaneously in mice. However,
    neoplasia of the pituitary gland has been associated with the
    administration of estrogen to certain strains of mice (Gardner, 1941),
    and it is considered to be associated with hormonal imbalance
    resulting from the administration of estrogenic hormones (Gardner,
    1941, 1948).

         In the estradiol-17 group, the estrogenic effects were more
    marked than those observed in mice treated with zeranol. These effects
    included brown degeneration in the adrenals, increased trabeculation
    in the sternum, conversion of male submaxilliary salivary glands to
    the female type, shrinkage of seminal vesicles, scarcity of secondary

    follicles and corpora lutea in the ovaries, uterine inflammation,
    endometriosis and hyalinization, cervical adenosis, vaginal epithelial
    keratinization, and mammary gland development. Tumourgenic effects in
    the positive controls included anterior lobe tumours of the pituitary
    gland and testicular interstitial cell tumours in males and mammary
    gland carcinomas with metastasizing adenocarcinomas in females
    (Everett,  et al., 1987a).

    Rats

         Four groups of 50 male and 50 female Sprague-Dawley rats each
    were maintained on diets containing 0 (negative control), 0.25, 2.5,
    or 25 ppm zeranol for 104 weeks. Two other groups of 25 male and 25
    female rats each, which served as positive dietary controls, were
    maintained on diets containing either 2.5 ppm estradiol-17 throughout
    the study or 25 ppm estradiol-17 for 8 weeks, which was reduced to
    2.5 ppm for the rest of the experiment (toxicity was observed at
    25 ppm). In another group of 25 females, which served as an implant
    positive control group, each animal was implanted s.c. with
    approximately 15 mg estradiol-17 at the commencement of the study.

         Haematology studies on 10 males and 10 females per group were
    carried out at weeks 26, 52, and 78 of the study and on all surviving
    rats at 104 weeks. Haematology, clinical chemistry analyses, and
    urinalyses were carried out on 15 males and 15 females per group
    (where available) at week 103 of the study. At week 104, all surviving
    animals were killed and autopsied. Full histopathological examinations
    were performed on all control, highest-dose zeranol, and estradiol-17
    test animals. Partial histopathological examinations (adrenals,
    cervix, ovaries, pituitary, prostate, seminal vesicles, testes, skin,
    kidneys, liver, lungs, mammary gland, and masses) were carried out on
    all other test animals. Animals dying during the course of the study
    were autopsied and complete histological examinations were performed
    on them.

         Male rats in the highest-dose zeranol group showed a slight
    decrease in weight gain compared to negative controls during the first
    52 weeks of the study, but weight gains were similar to controls
    during the remainder of the study. A slight decrease in body weight
    was observed in males in the 0.25 ppm group and in females in the 0.25
    and 2.5 ppm groups. Females in the highest-dose group showed similar
    weight gains to controls, except during the first 90 days of the
    study, when a reduction in body weight was observed. In contrast, rats
    receiving estradiol-17 showed a marked reduction in body weight, the
    greatest depression being noted in females implanted with
    estradiol-17. Levels of food consumption in the negative control and
    groups fed zeranol were similar. There was some reduction in food
    intake in rats in the positive control groups.

         Haematological studies showed that female rats in the 0.25 ppm
    zeranol group had increases in red blood cell parameters (haemaglobin
    level and red blood cell count) and white blood cell counts compared
    to controls. Animals in all other zeranol test groups had results
    similar to negative controls. Female rats in the positive dietary
    control groups showed elevated white blood cell counts, whereas those
    implanted with estradiol-17 showed decreases in red blood cell
    parameters and increased neutrophil counts. There were no significant
    inter-group differences in clinical chemistry or urinalysis results.

         Survival of test animals was similar in the negative control and
    zeranol test groups. In the positive dietary control groups, survival
    was lower than in the zeranol groups, but most deaths occurred after
    80 weeks of treatment. In the group implanted with estradiol-17,
    deaths occurred after 20 weeks, and there were no survivors at week 44
    of the study. No common causes of death were noted in the treated
    animals receiving zeranol, although those receiving estradiol-17
    showed a higher incidence of enlarged pituitaries than did controls.

         At autopsy, organ weight analyses of rats fed zeranol showed no
    significant differences between male rats and their respective
    controls. There was an apparent increase in uterus weight in females
    in the highest-dose group, but because of the large variation in ovary
    weights in this group, this was not statistically significant. Male
    rats receiving dietary estradiol-17 had significantly-decreased
    kidney weights, and females had reduced absolute ovary weights
    compared to controls.

         Histological examination of the tissues of the zeranol-treated
    animals showed no compound-related increases in neoplasms.
    Non-neoplastic findings in female rats included an increase in
    dilatation of the uterus in the highest-dose group, as well as an
    increase in stratified squamous epithelial cells of the cervix in the
    2.5 and 25 ppm groups. There was an increase in mammary hyperplasia in
    the 2.5 ppm group, but this was not statistically significant when an
    age-adjustment analysis was carried out. Kidney tubular epithelial
    haemosiderosis occurred more frequently in the 0.25 ppm test group
    than in controls, but this was not considered to be a compound-related
    effect.

         All females treated with estradiol-17 showed marked estrogenic
    effects, with significant changes in the ovaries, uterus (including
    squamous metaplasia and epithelial dysplasia), vagina and cervix, and
    mammary glands. Effects were also reported in the spleen, sternum,
    stomach, kidney, and lungs. In males treated with estradiol-17,
    effects were observed in the liver, pancreas, mammary gland, heart,
    testes, kidneys, and spleen. Histopathological changes in animals
    treated with estradiol-17 implants were more severe than those in
    animals receiving estradiol-17 in the diet. There was also a marked
    increase in pituitary adenomas (type 3) in test animals in the implant
    group compared with controls (Everett,  et al., 1987b).

    Special study on estrogenic potency

         The uterotropic response in sexually immature rats was used to
    compare the estrogenic potency of zeranol and its metabolites
    zearalanone (the 7-keto oxidation derivative) and taleranol
    (7--hydroxy epimer of zeranol) with estradiol-17. Administration of
    the test compound by the oral route showed the estrogenic potency of
    zeranol, zearalanone, and taleranol to be 1/150, 1/400, and 1/350,
    respectively, that of estradiol-17. By the s.c. route zeranol was
    1/500 less estrogenic than estradiol-17 (Everett  et al., 1987c).

    Special studies on no-hormonal effect levels

    Monkeys

         Eighteen female cynomolgus monkeys were bilaterally
    ovariectomized. After an 11-week stabilization period, the animals
    were assigned to 3 groups. The test groups were then dosed orally with
    zeranol in 0.2% carboxymethylcellulose in a flavoured beverage at dose
    levels equivalent to 0.05, 0.5, or 5 mg/kg b.w./day for 13 weeks. A
    control group was not used. Routine clinical chemistry, haematology,
    and urinalysis determinations were made one month prior to dosing, and
    at 1 and 3 months during the test period. Serum levels of insulin and
    thyroid stimulating hormone (TSH) were determined once prior to
    dosing, and at 1 and 3 months during the dosing period. FSH was
    determined on a weekly basis during the study through 4 weeks
    post-dosing.

         Clinical chemistry, haematology, and urinalysis parameters were
    within the normal ranges. No compound-related effects were observed on
    serum levels of insulin or TSH. FSH values during the 24 hours after
    the first daily dose of zeranol showed some variation, but no biphasic
    response was observed. FSH values measured during the course of the
    study showed variations, but no consistent depression was observed.

         Baseline measurements and measurements of maturation index of
    vaginal epithelial cells during the course of the study indicated an
    estrogenic effect in animals receiving 0.5 and 5 mg/kg b.w./day
    zeranol. The effect was not apparent at 0.05 mg/kg b.w./day
    (Singh  et al., 1984a; CIC, 1985).

         Three groups of 6 mature male cynomolgus monkeys each were dosed
    daily with zeranol at levels equivalent to 0.05, 0.5, or 5 mg/kg
    b.w./day for a period of 13 weeks. Routine clinical chemistry,
    haematology, and urinalysis determinations were made in each of the
    animals twice prior to dosing, then after 1 and 3 months of dosing.
    Serum levels of insulin, FSH, and testosterone were also measured. At
    the end of the dosing phase, testicular biopsies were taken and
    examined microscopically.

         Body-weight gain was normal during the test period. Clinical
    chemistry, haematology, and urinalysis parameters were within normal
    limits. Serum levels of insulin, TSH, and FSH showed no compound-
    related changes. Testosterone levels were highly variable in
    individual animals, but they showed no compound-related effects.
    Examination of testicular biopsies showed no effects on the
    spermatogonia, the primary and secondary spermatocytes, or the sperm.
    The Leydig cells were normal (Singh  et al., 1984b).

         Five groups of 6 sexually mature adult intact female cynamologus
    monkeys each were used for this study. Three groups were administered
    zeranol orally on a daily basis for 3 menstrual cycles at dose levels
    equivalent to 0.05, 0.5, or 5 mg/kg b.w./day. One group was dosed with
    the vehicle (0.2% carboxymethycellulose in a fruit flavoured drink)
    and the other with estradiol-17 at a dose level equivalent to
    0.01 mg/kg b.w./day. Haematology, clinical chemistry, and urinalysis
    determinations were made prior to dosing, during the dosing phase, and
    during a withdrawal period. Serum levels of insulin, TSH, FSH,
    estradiol-17, and progesterone were measured during the course of the
    study (every third of the menstrual cycle). Vaginal cornification
    (maturation index) and daily observations were made for changes in sex
    skin coloration or swelling.

         No treatment-related effects on body weight were observed. Sex
    skin changes during the cycles of dosing and withdrawal showed no
    compound-related effects in animals treated with zeranol or
    estradiol-17. None of the treatments had a significant effect on
    clinical chemistry or haematology parameters or on serum levels of
    insulin or TSH. There were no compound-related effects on serum levels
    or cyclic behaviour of FSH, estradiol, or progesterone in any of the
    test groups. There was no significant change in the type of vaginal
    epithelial cells during the dosing phase of the study (Singh  et al.,
    1984c; Singh & Griffin, 1984).

         Three groups of 8 mature female rhesus monkeys each were dosed
    daily with zeranol at levels equivalent to 0.5, 5, or 50 mg/kg
    b.w./day for three menstrual cycles, or 111 days, after a pretreatment
    control cycle. A control group of 6 monkeys received the vehicle
    (orange juice) used to suspend the zeranol. Daily blood samples were
    taken from each animal during the last pretreatment control cycle,
    every-other-day during the first two treatment cycles, and daily
    during the treatment cycle. Serum concentrations of estradiol,
    progesterone, LH and FSH were determined by radioimmunoassay.

         The average menstrual cycle showed no significant compound-
    related effects in the 0.5 or 5 mg/kg b.w./day group. By contrast, the
    menstrual cycle in the high-dose group was completely inhibited during
    the first 60 days of treatment, and markedly inhibited during the
    third cycle. Ovulation rates were similar in the control, low-dose,
    and mid-dose groups, but markedly inhibited in the high-dose group. No

    differences were observed in the quantitative pattern of estradiol
    secretion in the control and mid-dose groups; however, the pattern was
    altered in the high-dose group, where estradiol levels were
    significantly reduced. LH and FSH levels and patterns of secretion
    were similar to those observed in the pretreatment cycle in the
    control, low-dose, and mid-dose groups, but levels were high in the
    high-dose group, and the pattern of secretion was different than in
    the other groups (Hess, 1986).

    Special studies on mutagenicity

         The available mutagenicity data on zeranol and its metabolites,
    zearalanone and taleranol, are summarized in Tables 2, 3, and 4.

    Special studies on reproduction

    Rats

         Groups of 10 male and 10 female Sprague-Dawley rats, weighing
    approximately 200 g each that were 7 weeks of age at the beginning of
    the study, were maintained on diets containing 0, 0.25, 1.77, 12.5, or
    25 ppm zeranol commencing 4 weeks prior to mating and continuing until
    termination of the study. The offspring (F1 generation) were
    maintained on the test diets for 3 to 4 weeks after weaning. F0
    males and females were killed and necropsied after their respective
    functions were completed. The F1-generation animals were killed and
    necropsied at approximately 6 weeks of age.

         The F0 males showed a reduction in body weight compared to
    controls at all dose levels (19% in the high-dose group). Food
    consumption of males fed zeranol was also reduced. Although F0
    females fed zeranol showed only a slight reduction in food intake
    compared to controls, those in the 12.5 and 25 ppm groups showed
    significant reductions in body weight (21% in the high-dose group).
    Indices of fertility and general reproduction were evaluated using the
    fertility index, gestation index, live birth index, viability index
    (days 0 - 4), and lactation index (days 4 - 21). There were no
    significant dose-related effects. Male and female pups in the
    high-dose group showed a significant reduction in body-weight gain
    during the lactation period, and this was maintained during the
    post-weaning period. Decreased weight gain was also observed in the
    12.5 ppm group, but not in the lower-dose groups.

         At autopsy, absolute and relative organ weight analyses of the
    F0-generation animals showed decreased liver weight in all test
    groups. All males fed zeranol showed reduced absolute and relative
    seminal vesicle weights, and females had reduced absolute and relative
    ovary weights. Gross necropsy of the animals from both generations did
    not show any compound-related effects (Everett & Perry, 1984).

        Table 2.  Results of mutagenicity assays on zeranol
                                                                                              

    Test system       Test object        Concentration       Results     Reference
                                                                                              

    Ames test1        S. typhimurium     1 - 500 g/plate    Negative    Bartholomew & Ryan,
                      TA98, TA100,                                       1980
                      TA1538

    Ames test2        S. typhimurium     1 - 10000 g/       Negative    Jagannath, 1982a
                      TA100              plate

    Forward           Mouse lymphoma     25 - 600 g/ml      Negative    Cifone, 1982
    mutation          L5178Y TK+/-
    assay1            -3.7.2C cells

    Bone marrow       CD-1 mice          0.5, 1.5, 5 g/kg    Negative    Cimino, 1982
    cytogenic
    assay

    Hepatocyte        Adult male         1.3  10-3 to       Negative    Williams, 1983
    primary           F344 rats          1.3  10-5 mg/ml
    culture DNA
    repair assay

    DNA binding       Rat hepatocyte     Not available       Negative    Williams, 1984
    assay             primary
                      cell culture

    Rec-assay         B. subtilis        Not available       Positive    Scheutwinkel et al.,
                      H17, M45                                           1986

    SOS-chromo        E. coli            Not available       Negative    Scheutwinkel et al.,
    test2             PQ37                                               1986

    V79/SCE           Chinese hamster    Not available       Negative    Scheutwinkel et al.,
    test2             V79 cells                                          1986
                                                                                              

    1    With rat liver S-9 fraction.
    2    Both with and without rat liver S-9 fraction.

    Table 3.  Results of mutagenicity assays on zearalanone1
                                                                                              

    Test system    Test object         Concentration          Results      Reference
                                                                                              

    Ames test2     S. typhimurium      50, 500, 1000 g/      Negative     Ingerowski et al.,
                                       TA98, TA100, plate                  1981
                                       TA1535, TA1537
                                       TA1538

    Ames test3     S. typhimurium      1.0 - 10000 g/        Negative     Jagannath, 1982b
                                       TA98, TA100, plate
                                       TA1535, TA1537
                                       TA1538

    Forward        Mouse lymphoma      3.13 - 300 g/ml       Negative     Cifone, 1983
    mutation       L5178Y TK +/-
    assay          cells

    Bone marrow    CD-1 mice           0.5, 1.67,             Negative     Cimino, 1983
    cytogenic                          5 g/kg
    assay

    Hepatocyte     Adult male          5  10-10 to           Negative     Williams, 1985a
    primary        F344 rats           5  10-4 mg/ml
    culture DNA
    repair assay
                                                                                              

    1    7-keto oxidation derivative of zeranol.
    2    With rat liver S-9 fraction.
    3    Both with and without rat liver S-9 fraction.
        Special studies on teratogenicity

    Mice

         Pregnant CDI-1 mice were injected s.c. with log doses of zeranol
    ranging between 10 and 10000 ug/kg b.w./day on days 6 - 10 of
    gestation. Half of the mice were allowed to deliver, and the remaining
    half were delivered by caesarian section. At doses of 1000 and above
    the number of litters decreased from 81% in the controls to 35% in the
    treated animals. A decrease in the average weight of the pups and an
    increased number of stillborn litters were observed. Limited
    macroscopic examination showed increased medullary trabeculae in the
    bones (Davies  et al., 1977).

    Rats

         Groups of 4 female pregnant rats received zeranol orally in
    single doses of 4 mg on days 1, 2, 3, 4, and 5 of gestation, 1 mg/day
    during days 1 - 4 of gestation, or a single dose of 8 mg on day 6 of
    gestation. The rats were killed on day 9 of gestation. Zeranol
    appeared to inhibit implantation up to day 4 of administration. In
    experiments in which implantation occurred, the fetuses had begun to
    resorb by day 9 (IMC, 1980a).
        Table 4.  Results of mutagenicity assays on taleranol1
                                                                                              

    Test system      Test object         Concentration        Results      Reference
                                                                                              

    Ames test2       S. typhimurium      1 - 500 g/plate     Negative     Bartholomew & Ryan,
                     TA98, TA100                                           1980
                     TA 1548

    Ames test3       S. typhimurium      1 - 5000 g/plate    Negative     Jagannath, 1983
                     TA100

    Forward          Mouse lymphoma      20 - 160 g/ml       Borderline   Cifone, 1985
    mutation         L5178Y TK+/-
    assay2           -3.7.2C cells

    Bone marrow      ICR mice            8.5 - 85 mg/ml       Negative     Ivett, 1985a
    cytogenic
    assay

    Hepatocyte       Adult male          2  10-5 to          Negative     Williams, 1985b
    primary          F344 rats           2  10-1 mg/ml
    culture DNA
    repair assay

    Chromosomal      Chinese             12.5 - 250 g/ml     Positive     Ivett, 1985b
    aberration       hamster ovary                            (- act.)
    frequency        cells                                    Negative
    assay3                                                    (+ act.)

    Dominant         H/a (ICR)BR         0.5, 1.5, 5.0        Negative     Brusick & Myhr,
    lethal assay     mice                g/kg b.w./day                     1986
                                                                                              

    1    7-hydroxy epimer of zeranol.
    2    With rat liver S-9 fraction.
    3    Both with and without rat liver S-9 fraction.
    
         A three-generation study in rats was performed in which males
    weighing 69 - 113 g each and females weighing 65 - 109 g each were fed
    zeranol in the diet at levels of 0, 0.01, 0.10, or 0.20 ppm for each
    successive generation, each comprising three consecutive litters.
    Animals in each generation received zeranol in the diet for 70 days
    prior to mating, they were removed from the diet from day of delivery
    of the dams throughout lactation until weaning, and then they were
    reinstated at the same levels as at the start of the study. Pups from
    the Fb litters were used for the reproduction of the subsequent
    generation; the pups from the Fc litters were used for the
    teratogenicity study. While there were some differences in the litter
    data for the three consecutive generations which appeared to be
    significant, there was no trend or dose-response relationship. No
    adverse effects were observed which could be attributed to treatment
    with zeranol (Wazeter & Goldenthal, 1976).

         Zeranol, administered orally at doses of 0.312, 1.25, or
    5.0 mg/kg b.w./day to male rats for 60 days prior to mating with
    untreated females, induced delayed insemination upon cohabitation and
    delayed initiation of conception in the females (Williams, 1982).

         Zeranol, administered orally at doses of 0.312, 1.25, or
    5.0 mg/kg b.w./day to female rats for 60 days prior to mating with
    untreated males, caused a decrease in the litter size, an increase in
    stillbirths, and a decrease in neonate survival (Williams, 1982).

         Pregnant female rats were administered orally 2 or 6 mg/kg
    b.w./day zeranol from days 6 - 15 of gestation. At laparotomy on day
    20 of gestation, reduced numbers of live fetuses and increased numbers
    of resorptions were noted (Williams, 1982).

    Rabbits

         Zeranol was administered orally at doses of 1 or 5 mg/kg b.w./day
    to pregnant does on days 6 - 18 of gestation. Fetuses were removed by
    laparotomy on day 28. No abnormalities were noted (Williams, 1982).

    Acute toxicity
                                                                        

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

    Mouse      ?           oral         > 40             IMC, 1980b
               F           i.p.         4.4              IMC, 1980b

    Rat        ?           oral         > 40             IMC, 1980b
               F           i.p.         9-11             IMC, 1980b
                                                                        

         Groups of 25 rats/sex/dose received by oral gavage 0 or
    200 mg/kg b.w. zeranol, and they were sacrificed 24 hours after
    dosing. There was a decrease in blood glucose levels in all treated
    rats, and an increase in total cholesterol in the males and a decrease
    in the females. There was a decrease in the weight of the epididymis
    of the males, and an increase in uterine weight in the females (Albany
    Medical Collage, 1980).

    Short-term studies

    Mice

         Groups of 20 CD-1 mice equally divided by sex were maintained on
    diets containing 0, 1, 5, 25, 50, or 100 ppm zeranol for 8 weeks. No
    deaths or clinical effects were observed during the course of the
    study. There were no significant differences in body weights, food
    consumption, or water consumption between test and control groups. At
    autopsy, no compound-related gross lesions were observed. No
    compound-related differences were observed in absolute or relative
    organ weights between test and control groups of the liver in males
    and females or of the testes in males. However, changes were observed
    in the absolute and relative weights of the ovaries (decreased) at
    dose levels of 25, 50, and 100 ppm zeranol and of the uterus
    (increased) at 100 ppm zeranol in females (Perry & Everett, 1984).

    Rats

         Groups of 40 Sprague-Dawley rats equally divided by sex were
    maintained on diets containing the equivalent of 0, 0.02, 0.18, 1.2,
    or 8.8 mg/kg b.w./day zeranol for a period of 13 weeks. Body weights
    and food consumption were determined weekly. Haematological, clinical
    chemistry, and urinalysis studies were carried out at weeks 5 and 13.
    Ophthalmoscopy was carried out at week 10. At termination of the
    study, gross observations and organ weight determinations were made on
    all animals, and histopathological examinations were performed on the
    principal organs and tissues from 10 animals of each sex in the
    high-dose and control groups.

         Six deaths occurred during the course of the study, which did not
    appear to be compound-related. Only minor decreases in food
    consumption were observed in the test groups compared to the control.
    However, there was marked reduction in weight gain compared to
    controls in the high-dose group, particularly in males. No compound-
    related changes were observed in haematological, clinical chemistry,
    or urinalysis parameters. Organ-weight analysis showed a slight
    increase in relative liver weights in both males and females and in
    the kidneys of rats in the high-dose group. Histological examination
    showed an increased incidence of vacuolation in hepatocytes from both
    males and females in the high-doss group as well as fat accumulation

    in these cells. The kidneys of males in the high-dose group showed a
    significant increase in calcified casts. These effects were only
    marginal in the 1.2 mg/kg b.w./day group. No compound-related effects
    were observed at 0.18 mg zeranol/kg b.w./day (Everett  et al., 1983).

         Twenty intact juvenile female rats were implanted s.c. with 12 mg
    pellets of zeranol and sacrificed after 150 days. Five control animals
    were used. Decreased growth, cysts in the ovaries, mammary glands, and
    other parts of the body, decreased ovarian and uterine weights,
    absence of corpora lutea, and marked squamous metaplasia of the
    uterine mucosa were noted (Huis in't Veld & Kroes, 1974).

         Groups of 30 - 35 rats/sex/group received by oral gavage 0 or
    200 mg/kg b.w./day zeranol for 4 days. There were decreases in total
    cholesterol and blood glucose levels, decreases in weights of the
    testes, seminal vesicles, and epididmes of males, increases in weights
    of the adrenals of males, and increases in weights of the uterus and
    adrenals of females. Microscopically, the ovaries showed prominent
    corpora lutea, and the testicular tubules contained spermatogonia but
    no adult or mature spermatozoa. The lymphoid tissue contained a number
    of large multinucleated cells (Albany Medical College, 1980).

         Groups of 5 albino Manor Farm rats/sex/dose received by oral
    gavage 25, 50, 100, 200, or 400 mg/kg b.w./day zeranol 5 days/week for
    6 weeks. A control group was not included. During the 4th week of the
    study, the 100 mg dose was increased to 800 mg/kg b.w./day and the
    200 mg dose was increased to 1600 mg/kg b.w./day. Effects were
    observed in all treatment groups that included changes in external
    genitalia (reduction in the size of the testes in males and
    enlargement of the vulva in females). Irritability, flaccidity,
    alopecia, excessive micturition, and reduced growth rate were also
    noted. After microscopic examination, changes noted were spermatogenic
    arrest, atrophy of the prostate, seminal vesicles, and coagulating
    glands, absence of graafian follicles, endometrial hyperplasia,
    squamous metaplasia of the seminal vesicles, and calcified casts in
    the kidneys (IMC, 1980c).

         Three groups (number not given) of male and female Sprague-Dawley
    rats received s.c. 0, 2.25, or 9.0 mg/kg b.w./day zeranol for 14 days.
    The growth rate was reduced in the treated animals. There were
    decreases in the relative weights of the gonads, prostate, and thymus
    in males and of the gonads and thymus in females. Additionally, there
    were increases in the relative weights of the seminal vesicles and
    adrenals in males and of the uterus and adrenals in females. A
    significant reduction in the number of corpora lutea was also noted
    (IMC, 1980d).

         Groups of male and female rats received 0.25, 1.25, or 6.25 mg/kg
    b.w./day zeranol in the diet for 13 weeks. Slight growth suppression
    at the two highest doses was observed (Williams, 1982).

         Groups of male and female rats were administered 0.1, 0.8, or
    6.4 mg/kg b.w./day zeranol for 26 weeks. Reduced food intake and
    body-weight gains, a tendency toward lower haemoglobin values in
    males, and mild hepatic changes, including irregularity in hepatic
    cell size, were noted in females at the highest dose (Williams, 1982).

    Dogs

         One dog/sex/dose received orally by capsule 25, 50, 100, 200, or
    400 mg/kg b.w./day zeranol 5 days/week for 6 weeks. During the 4th
    week of treatment the 100 mg dose was increased to 800 mg/kg b.w./day
    and the 200 mg dose was increased to 1600 mg/kg b.w./day. No control
    animals were included. Beginning with the second week of treatment,
    all females showed swollen vulvae. All males showed a decrease in
    testicular size. At a dose of 100 mg/kg b.w./day and above, males and
    females showed slight to marked increases in leucocytes and relative
    decreases in lymphocytes. At 200/1600 mg/kg b.w/ day, there were
    slight decreases in haemaglobin and haematocrit values, and increases
    in erythrocyte sedimentation rates. Microscopically, mammary ductal
    proliferation was noted in both males and females, vulval swelling
    with cornification of vaginal epithelium and ovarian atrophy was
    observed in females, and atrophy of the seminiferous epithelium and
    prostate and hyperplasia or squamous metaplasia of the prostatic
    urethra were noted in males. At doses of 400 mg/kg b.w./day and above,
    there were increases in bone marrow cellularity and in the
    myeloid/erythrocyte ratio (Williams, 1982).

         Groups of male and female dogs received orally by capsule 0.25,
    1.25, or 6.25 mg/kg b.w./day zeranol for 14 weeks. Following the
    thirty-first dose, the 0.25 mg dose was increased to 12.5 mg/kg
    b.w./day. There was a trend toward a reduction in the size of the male
    sex organs and an increase in uterine weight. Microscopically, there
    was an arrest in spermatogenesis, and prostatic epithelial atrophy was
    seen in one male at 12.5 mg (Williams, 1982).

         Groups of male and female dogs were fed diets containing 10, 100,
    or 1000 ppm zeranol for 29 weeks. At 1000 ppm, noted changes included
    weight loss in one male, rapid sedimentation rates, reduced
    haemoglobin, increased leucocytes, and reduced lymphocytes in 3 males.
    Microscopically, changes noted in the 1000 ppm group included
    testicular atrophy, squamous metaplasia of the prostate, endometrial
    hyperplasia, and ovarian atrophy. Hypercullularity of the bone marrow
    and slight squamous metaplasia of the bladder were also noted
    (Williams, 1982).

    Long-term studies

    Rats

         Groups of 25 - 35 male and female rats were fed 0, 0.1, 0.8, or
    6.4 mg/kg b.w./day zeranol for one year. During the 36th week of the
    study the 0.1 mg/kg b.w./day dose level was increased to 20 mg/kg
    b.w./day. Body-weight gain was depressed in all the treated animals.
    The rats in the 0.1/20 mg/kg b.w./day group showed depressed
    haemoglobin, increased thrombocyte counts, increased prothrombin time,
    decreased absolute and relative weights of the prostate, ovary, and
    seminal vesicles, and increased absolute and relative weights of the
    uterus and pituitary. Hepatic cell glycogen depletion and irregularity
    in hepatic cell size were observed in male rats only. Nephron
    degenerative changes, slight to moderate atrophy of the ovary, seminal
    vesicles, and prostate, slight reduction in the number of mature
    spermatozoa, and instances of hypocellularity of the bone marrow were
    also noted in this group of animals. Marked degenerative changes were
    seen in the livers of the animals which died during the study
    (Williams, 1982).

         Groups of 25 Charles River CD rats/sex/dose were fed 0, 0.1, 0.8,
    or 6.4 mg/kg b.w./day zeranol for 104 - 105 weeks. During the 28th
    week of the study the 0.1 mg/kg b.w./day dose level was increased to
    20 mg/kg b.w./day. Body-weight gain was depressed in all the treated
    animals in the 6.4 and 0.1/20 mg/kg b.w./day dose groups. Greater
    frequency of alopecia was observed in the two highest dose groups, and
    one rat in the 6.4 and 4 rats in the 0.1/20 mg/kg b.w./day dose groups
    developed cataracts during the last quarter of the study. Haemoglobin
    and haematocrit values were reduced at 0.1/20 mg/kg b.w./ day, which
    was more severe in females than in males. The animals in the
    0.1/20 mg/kg b.w./day group had lower absolute weights of the seminal
    vesicles, testes, ovaries, and prostate. Macroscopically, the findings
    were those commonly noted in aging animals. Microscopically, changes
    were seen in the liver, ovaries, uterus, testes, prostate, and seminal
    vesicles of all the rats treated with zeranol. The changes in the
    liver included hepatic cell depletion, irregularity in hepatic cell
    size, parenchymal cell vacuolation, chronic inflammatory cell
    infiltrates, and occasional instances of nodular hyperplasia; in the
    ovaries, prostate, seminal vesicles, and testes, slight to moderate
    atrophy was noted; in the uterus the observed changes included
    endometritis, squamous metaplasia, and cystic hyperplasia of the
    endometrium (Woodard, 1968).

    Dogs

         Groups of male and female dogs were fed 0.025, 2.5, or 25 mg/kg
    b.w./day zeranol for one year. Slight testicular atrophy, moderate
    prostatic atrophy, and squamous metaplasia were observed at the middle
    dose. At the high dose, observed changes included increased
    thrombocytes, decreased lymphocytes, rapid sedimentation rates,
    elevated leucocyte counts, and marked cornification of the vaginal
    epithelial cells. At terminal sacrifice, treatment-related changes in
    the 25 mg/kg b.w./day group included marked atrophy of the gonads,
    squamous metaplasia and inflammatory changes in the prostate and
    uterus, squamous metaplasia of the urinary bladder epithelium, an
    apparent increase in the myeloid elements of the bone marrow, and
    endometrial hyperplasia (Williams, 1982).

         Groups of 4 beagle dogs/sex/dose were fed 0, 1, 100, or 1000 ppm
    zeranol in the diet for 104 weeks. Body weights of the male dogs in
    all treatment groups were slightly higher than those of the controls.
    Most of the observed changes were noted at the highest dose.
    Haematological changes included rapid sedimentation rates, high
    leucocyte counts, and reduced lymphocytes. Increased relative weights
    of the prostate and pituitary and decreased relative weights of the
    gonads in males, increased uterine relative weights in females, and
    increased relative weights of the liver, kidneys, adrenals, and
    thyroid of both males and females at the highest dose were noted.
    Macroscopically, observed changes included enlarged mammary glands,
    small testes, edematous prepuces, and enlarged vulvae, prostate, and
    uteri. Microscopically, marked atrophy of the gonads, especially the
    ovaries (which were reduced to cysts), squamous metaplasia and
    inflammatory changes in the uterus and prostate, and chronic
    inflammatory changes in the bladder were observed. Two males and one
    female in the 100 ppm group had varying degrees of myeloid hyperplasia
    and one male had marked atrophy of the bone marrow (Woodard, 1968).

         Groups of 16 female beagle dogs were administered orally by
    capsule 0, 15, or 38 mg/kg b.w./day zeranol in a cyclic manner
    (21 consecutive days of dosing followed by 7 days off the drug) for a
    period of seven years (91 cycles). Two high-dose and two low-dose dogs
    died during the third year, and 3 low-dose dogs died or were
    sacrificed in a moribund state during the fourth year of the study.
    Clinical signs of toxaemia and gross lesions associated with
    pyometritis were noted in these dogs. To prevent further deaths
    related to pyometritis all surviving dogs were hysterectomized at
    3 - 3.5 years into the study. Prior to hysterectomy, an increased
    incidence of anorexia and lethargy were noted in the treated animals.
    Mean body weights were slightly lower than controls in the 15 mg/kg
    b.w./day group; they were significantly lower than controls in the
    high-dose group. Haematological changes included a slight but
    consistent reduction in absolute and relative adrenal weights in the

    animals in the low-dose group at the four year interim sacrifice. At
    terminal sacrifice, significant increases in mean and relative ovarian
    weights of the animals in both treated groups were noted. Zeranol had
    a relatively strong uterotropic effect and caused a sharp increase in
    the proliferation and cornification of the vaginal, cervical, and
    vulvar mucosa, cystic endometrial hyperplasia, endometriosis interna,
    and pyometritis (Hogan, 1981a).

    Monkeys

         Groups of 16 sexually mature female rhesus monkeys (Macaca
    mulatta) were administered orally by gavage 0, 15, or 75 mg/kg
    b.w./day zeranol in a cyclic manner (21 days of consecutive dosing and
    7 days off the drug) for a period of 10 years (131 cycles). Control
    animals were administered the vehicle, which was a mixture of ethanol
    and methylcellulose. Interim sacrifice was performed at one year
    (2 animals) and at 2 and 4 years (4 animals each).

         Seven animals died during the first nine months of causes
    unrelated to treatment and were replaced to maintain the number of
    animals per group. Ophthalmological changes included bilateral
    hypopigmented foci in the macular and perimacular regions of the
    retina, grainy appearance of the macula, and diminution or loss of the
    macular reflex. Palpable mammary nodules and/or enlarged axillary
    lymph nodes were seen sporadically in some of the animals. Palpable
    uterine masses were observed in the high-dose animals at a markedly
    higher incidence than in the control and low-dose groups. Prolonged
    periods of amenorrhea were observed in the high-dose animals, which
    was followed, in later years of the study, by prolonged periods of
    vaginal bleeding. The treated monkeys gained less weight than the
    controls throughout the study; at termination the mean body weight of
    females at 15 mg/kg b.w./day was 15% lower, and that of females at
    75 mg/kg b.w./day was 25% lower, than body weights of controls.

         During the first 1.5 years of the study there were transient
    reductions in mean haemaglobin concentrations and haematocrit and
    erythrocyte values in the treated animals as compared to the controls.
    Sporadic increases in the measured clotting time were noted throughout
    the study. No other haematological changes were noted. Sporadic
    increases in SGPT activity were noted at 15 mg/kg b.w./day, while
    consistently higher SGPT activities were observed at 75 mg/kg
    b.w./day. A similar pattern was observed with serum triglyceride and
    cholesterol concentrations.

         The animals in the high-dose group had consistently higher mean
    absolute and relative weights of the liver and uterus and lower mean
    absolute and relative ovarian weights than the controls. Similar
    observations, but to a lesser degree, were noted in the animals fed
    15 mg/kg b.w./day zeranol. At terminal sacrifice, the mean relative
    weight of the pituitary and mean absolute and relative adrenal weights

    of the animals in the high-dose group were higher than those of the
    controls. No treatment-related macroscopic changes were noted at the
    1- and 2-year interim sacrifices. At A years, there were dose-related
    increases in the incidence and degree of endometrial proliferation. At
    10 years, treatment-related changes included enlarged uteri, thickened
    endometrium and myometrium, enlarged oviducts, and endometriosis
    externa.

         No treatment-related microscopic changes were noted at one year
    into the study. At two years, one benign adenoma was noted in one
    animal and a relative increase in mammary acinar tissue and duct
    development were noted at 75 mg/kg b.w./day zeranol. At 4 years,
    treatment-related changes in the high-dose group included endometrial
    hyperplasia with occasional cystically dilated glands (all 4 animals),
    absence of mature follicles and corpora lutea, superficial atrophy of
    the cervical epithelium with squamous metaplasia of the basilar
    portions of cervical glands, and increased lobular development and
    mild ductular epithelial proliferation. At 10 years, treatment-related
    changes were noted in most of the animals in the high-dose group that
    included hyalization of the subepithelial connective tissue in the
    vagina, squamous metaplasia of the basilar portions of the cervical
    glands, ovarian atrophy and absence of corpora lutea, and marked
    ductal and acinar hyperplasia of the mammary gland. Cystic endometrial
    hyperplasia, myometrial hypertrophy, and endometriosis externa were
    noted in all the animals at 75 mg/kg b.w./day zeranol and in three
    animals in the low-dose group. Leiomyomas of the uterus/cervix
    (fibroids) were present in 1/12, 2/7, and 2/14 animals in the control,
    low-, and high-dose groups, respectively (Hogan, 1981b).

    COMMENTS

         The toxicological data available to the Committee included
    results from the requested studies, as well as mutagenicity,
    reproduction, and teratogenicity data.

         Zeranol was shown to be a weak estrogen in long-term studies in
    mice, rats, dogs, and monkeys. Most of the changes noted occurred in
    mammary glands and organs of the reproductive tract. Zeranol did not
    cause changes in other reproductive parameters in rats, and was not
    teratogenic in mice or rats. Zeranol and its metabolites zearalanone
    and taleranol were not mutagenic in a number of tests in bacterial and
    mammalian systems. Zeranol (at an unspecified concentration) gave a
    positive result in the Rec-assay (Bacillus subtilis) and taleranol
    gave a positive result in the test with Chinese hamster ovary cells in
    the absence of activation, but a negative result with activation.

         In the carcinogenicity study performed in rats, dietary levels of
    zeranol up to 25 ppm (equivalent to 1.25 mg/kg b.w./day) had
    estrogenic but not carcinogenic effects. In the study performed in
    mice, zeranol had significant estrogenic effects in male mice in the
    highest dose group, receiving zeranol at 15 ppm in the diet
    (equivalent to 2.25 mg/kg b.w./day); these mice also showed a higher
    incidence of anterior lobe tumours of the pituitary gland than did
    mice in the negative control group. Such tumours rarely occur
    spontaneously in mice but are known to result from administration of
    estrogenic hormones. The positive control group receiving a diet
    containing 2.5 ppm estradiol-17 showed a higher incidence of anterior
    lobe tumours of the pituitary than did either animals receiving
    zeranol or animals in the negative control group. Thus the Committee
    concluded that the carcinogenic effect of zeranol was associated with
    its estrogenic properties, and that the determination of a
    no-hormonal-effect level for tumours would permit an estimate of safe
    levels of exposure to be made.

         A no-hormonal-effect level could not be established in male
    cynomolgus monkeys since no estrogenic effects were observed even at
    the highest dose administered (5 mg/kg b.w./day). In intact female
    cynomolgus and rhesus macaque monkeys, 5 mg/kg b.w./day was
    established as a no-hormonal-effect level. In ovariectomized female
    cynomolgus monkeys the no-hormonal-effect level was 0.05 mg/kg
    b.w./day. The Committee concluded that this model could be relevant to
    the human population and, since the ovariectomized female cynomolgus
    monkey is highly sensitive to estrogenic substances, adopted a
    conservative approach by using this study as a basis for setting an
    ADI for human beings.

    EVALUATION

    Level causing no hormonal effect

         Monkey:   0.05 mg/kg b.w./day.

    Estimate of acceptable daily intake

         0 - 0.5 g/kg b.w.

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    See Also:
       Toxicological Abbreviations
       ZERANOL (JECFA Evaluation)