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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION



    TOXICOLOGICAL EVALUATION OF CERTAIN 
    VETERINARY DRUG RESIDUES IN FOOD



    WHO FOOD ADDITIVES SERIES 45





    Prepared by the
    Fifty-fourth meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)



    World Health Organization, Geneva, 2000

    MELENGESTROL ACETATE

    First draft prepared by
    Professor Fritz R. Ungemach
    Institute of Pharmacology, Pharmacy and Toxicology, Veterinary
    Faculty, University of Leipzig, Leipzig, Germany

            Explanation 
            Biological data 
                Biochemical aspects 
                    Absorption, distribution, and excretion 
                    Biotransformation 
                Toxicological studies 
                    Acute toxicity 
                    Short-term studies of toxicity 
                    Long-term studies of toxicity and carcinogenicity
                    Genotoxicity 
                    Reproductive toxicity 
                        One-generation studies 
                        Developmental toxicity 
                    Special studies: Immunotoxicity 
                Observations in humans 
            Comments 
            Evaluation 
            References 


    1.  EXPLANATION

         Melengestrol acetate
    (17alpha-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione) is
    a synthetic progestogen which is active after oral administration. It
    is used to improve the efficiency of feed conversion, promote growth,
    and suppress estrus in female beef cattle. The range of approved doses
    is 0.25-0.50 mg/heifer per day. The drug can be administered alone or
    in combination with other growth-promoting drugs. Melengestrol acetate
    is fed for the duration of the fattening and finishing period, usually
    for 90-150 days. Melengestrol acetate has not previously been
    evaluated by the Committee.

         Most of the toxicological studies submitted to the Committee were
    conducted before 1979 according to the standards of those days and
    were not performed in compliance with good laboratory practice (GLP).
    The results of studies conducted more recently, according to
    appropriate standards for protocol and conduct, were consistent with
    those of the older studies. Some of the reports did not include data
    on individual animals and other important details, so that the
    conclusions could not be confirmed independently. The missing data
    were not, however, pivotal for the evaluation.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, and excretion

         Limited studies, which did not comply with GLP, of the
    pharmacokinetics of melengestrol acetate in cattle, rabbits, and
    humans have been reported. The database was limited, and important
    details such as the bioavailability of oral doses and plasma kinetics
    and studies in other laboratory species were missing. Melengestrol
    acetate labelled at the 6-methyl position with 3H or 14C was used
    in these studies. As this methyl group can be removed by oxidative
    attack during biotransformation, radiolabel might be lost through
    exhalation of 14CO2 (Cooper, 1967).

          Cattle 

         Four heifers were fed diets containing melengestrol acetate at a
    dose of approximately 0.5 mg/head per day for 4 months. Three animals
    then received 3H-melengestrol acetate for 21 days, and one received
    14C-labelled material for 7 days while housed in a metabolism stall.
    The radiolabelled compounds were administered in gelatine capsules.
    Urine obtained through a catheter and faeces were collected and
    assayed for radiolabel. About 72% of the 3H label was excreted, but
    the combustion method used resulted in low recovery of tritium. A
    similar excretion pattern was found in the one animal given
    14C-melengestrol acetate. The ratio of radiolabel excreted in faeces
    and urine was about 6:1. At the end of the experiment, the animals
    were slaughtered and the total radiolabel was measured in tissues and
    organs by liquid scintillation spectrometry. The highest concentration
    was found in bile (mean, 110 g/kg expressed as 3H-melengestrol
    acetate equivalents) and in the contents of the jejunum (4.4 g/kg)
    and large intestine (7.9 g/kg) (Krzeminski et al., 1981). These
    findings are consistent with the results of a previous study in
    heifers with cannulated bile ducts, which showed that bile was the
    main route of excretion (Neff, 1964). Another source of faecal
    melengestrol acetate in cattle is unabsorbed material, as shown in the
    study of Davis (1973) in which 10-17% of orally administered,
    unlabelled melengestrol acetate passed through the gastrointestinal
    tract without being absorbed. Analysis of melengestrol acetate in
    organs and tissues showed the highest concentrations of total
    radiolabel in the liver (mean, 12 g/kg as 3H-melengestrol acetate
    equivalents) and fat (7.7 g/kg). In excretory organs, the highest
    concentrations were found in the wall of the digestive tract (2-11
    g/kg), followed by salivary glands (3 g/kg) and kidney (1.6 g/kg).
    3H-Melengestrol acetate equivalents were detected at concentrations
    > 2 g/kg in mammary glands, oviducts, adrenal glands, and thymus but
    at about 1 g/kg (e.g. 0.7 g/kg in muscle) in all other tissues. The
    limit of detection was 0.5 g/kg as 3H-melengestrol acetate
    equivalents. 

          Rabbits 

         Two rabbits were given a single dose of
    [6-methyl-14C]melengestrol acetate acetate by gavage at 47
    mg/animal. Within 7 days, 59% of the administered radiolabel was
    excreted (15% in urine and 44% in faeces), with a peak rate of
    elimination on day 1 and < 0.1% on day 7 (Cooper, 1967). 

          Humans 

         14C-Melengestrol acetate was administered orally as a single
    dose to six women aged 34-57, at doses of 3.2-4.8 mg to three women
    and of 93.5-95.8 mg to the other three. As the preparations had
    different specific radioactivities, the amount of radiolabel
    administered was similar for all patients (1.11-3.01 Ci). Urine and
    faeces were collected for 3-7 days from women who received the low
    dose and for 5-12 days from those given the high dose. The rate of
    excretion of radiolabel declined sharply after day 1, and excretion
    was substantially complete within 10 days. The total radiolabel
    recovered from urine and faeces was 44-87% (mean, 74%). The rates of
    urinary excretion at the high and low doses were similar, but faecal
    excretion was slower at the low dose. The half-time was 3-5 days at
    the low dose and < 1 day at the high dose (Cooper, 1967; Cooper et
    al., 1967). 

    2.1.2  Biotransformation

         The biotransformation of radiolabelled melengestrol acetate in
    cattle, rabbits, and humans was determined in the studies described
    above and showed a high rate of metabolism with the formation of
    numerous metabolites. The metabolites have not been adequately
    identified or characterized with respect to their biological activity.

          Cattle 

         Fat, liver, kidney, and muscle from the four heifers treated
    orally with 3H- or 14C-melengestrol acetate after feeding of
    unlabelled melengestrol acetate (described above) were assayed for
    melengestrol acetate. The steroid was extracted sequentially with
    organic solvents and isolated by column chromatography, and the
    extracts were analysed by gas chromatography or liquid scintillation
    counting. Unchanged melengestrol acetate represented 75-86% of the
    total radiolabel in fat, 29% in liver, 48% in muscle, and 29% in
    kidney 6 h after treatment. The formation of metabolites of
    melengestrol acetate in liver was monitored by thin-layer
    chromatography during the extraction procedure, and radiolabel was
    measured in fractional plate scrapings (3H-melengestrol acetate) or
    by autoradiography (14C-melengestrol acetate). Three major peaks
    were seen, none of which exceeded 1 g/kg or 10% of the total
    radiolabel. These polar metabolites were not further identified. The
    uniform distribution of 3H2O in all tissues except fat indicates
    metabolic loss of tritium from the 6-methyl position and the formation
    of further metabolites (Krzeminski et al., 1981). In a recent study of

    bovine liver microsomes  in vitro, several hydroxylated metabolites
    of melengestrol acetate were separated and identified by
    high-performance liquid chromatography (HPLC) with mass spectrometry
    (MS) and showed a pattern of biotransformation similar to that in
    rats. The chemical structure of these metabolites was not reported
    (Metzler, 1999). In cattle, approximately 15% of a dose of
    melengestrol acetate was excreted intact in the urine, but no
    information on the nature of the urinary metabolites was available
    (Lauderdale, 1977a).

          Rats 

         The biotransformation of melengestrol acetate by Arochlor-induced
    rat liver microsomes  in vitro yielded seven monohydroxylated and
    five dihydroxylated metabolites, when separated by HPLC and identified
    by HPLC/MS, but no information on the chemical structure of the
    various metabolites was available (Metzler, 1999).

          Rabbits 

         Unconjugated steroids were extracted by chloroform from the urine
    of two rabbits receiving [6-methyl-14C]melengestrol acetate. The
    conjugated fraction was hydrolysed to yield the glucuronides and,
    subsequently, the sulfates. The subfractions of the various conjugates
    were further separated by partition column chromatography. Attempts
    were made to identify the main metabolite by physical measurements and
    microchemical reactions according to the standards of those days. Only
    44% of the total radiolabel excreted in urine was recovered in the
    unconjugated (14%) and conjugated fractions (30%). The conjugated
    steroids were mainly glucuronides, and only 4.4% were sulfates. The
    elution profile after column chromatography of the unconjugated
    extract and the hydrolysed glucuronides showed two major and numerous
    smaller peaks. One peak was identified as the 6-methyl-hydroxylated
    metabolite of melengestrol acetate,
    17-acetoxy-6-hydroxymethyl-16-methylenepregna-4,6-diene-3,20-dione),
    which is excreted in both the glucuronide and the unconjugated form.
    Another urinary monohydroxylated metabolite,
    17-acetoxy-2a-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione
    , was postulated but not measured. No attempts were made to identify
    the other radioactive peaks in urine or the radiolabelled material
    excreted in faeces (Cooper, 1967).

          Humans 

         The urine of four of six women treated orally with
    [14C-methyl]melengestrol acetate was processed for conjugated
    metabolites by conventional methods of hydrolysis, and 68% of the
    radiolabel recovered from urine was found to be conjugates of
    hydrophilic melengestrol acetate metabolites and 22% unconjugated
    steroids. About 25% of the conjugates were glucuronides and 14% were
    sulfates. The remaining hydrolysis residues were not identified. After
    chromatography of the free and conjugated urine fractions on a Celite
    column, 22 peaks were obtained which represented at least 13 distinct

    metabolites. One of the metabolites was identified as
    2a-monohydroxylated melengestrol acetate
    (17-acetoxy-2alpha-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-
    dione) by ultraviolet and infrared spectroscopy, re-chromatography on
    paper, and microchemical reactions. This metabolite was present in the
    conjugated and unconjugated forms and accounted for about 2% of the
    administered dose of 14C-melengestrol acetate. The
    6-methyl-monohydroxylated metabolite could not be detected. About 11%
    of the dose of radiolabelled melengestrol acetate was associated with
    the 12 remaining metabolites, which were not identified by their
    chemical structure but by their nature. Thus, all of the metabolites
    were assumed to contain the intact steroid nucleus of melengestrol
    acetate. One metabolite was less polar than melengestrol acetate,
    while the others were mono-, di-, or trihydroxylated derivatives, the
    polarity of at least seven of which suggested the presence of more
    than one hydroxyl group. Seven compounds showed the 4,6-dien-3-one and
    the 20-ketone form of the parent substance. Five of these also
    appeared to contain the 17alpha-acetate group, whereas the remaining
    two were apparently 17alpha-hydroxylated. No 21-hydroxylated
    metabolites were identified, although it was presumed that
    21-hydroxylation of at least one of the more polar metabolites might
    occur. In faeces, 35% of the radiolabel was unconjugated and 22% was
    conjugated. Chromatography of the unconjugated and hydrolysed
    conjugates showed the presence of unchanged melengestrol acetate.
    Attempts to characterize the hydrophilic conjugated metabolites were
    unsuccessful (Cooper, 1967; Cooper et al., 1967). 

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The acute toxicity of melengestrol acetate was studied in mice,
    rats, and rabbits by oral, dermal, subcutaneous, or intraperitoneal
    administration. None of the experiments complied with GLP, and the
    studies were described in abstract form only; the study design as well
    as the reports fell short of current standards. The results (Table 1)
    indicate that melengestrol acetate has low acute toxicity in rodents
    after oral or intraperitoneal administration, although the studies
    were limited by the large volume of vehicle that had to be
    administered. No deaths were observed in any study, and the only
    reaction reported was sedation. Dermal application of melengestrol
    acetate to intact or abraded skin of rabbits at the maximum achievable
    dose of 22 mg/kg bw caused no toxic reaction.

    2.2.2  Short-term studies of toxicity 

         In short-term studies of toxicity in mice, rats, rabbits, dogs
    and monkeys, melengestrol acetate had a greater effect in females than
    in males, with hormonal (progestational and corticosteroidal) effects
    as the most sensitive end-points.


        Table 1. Results of studies of acute toxicity with melengestrol acetate

                                                                                                          
    Species (strain)      Sex   Route             Vehicle            LD50         Reference
                                                                     (mg/kg bw)
                                                                                                          

    Mouse (NR)            NR    Intraperitoneal   NR                 > 2500       Webster et al. (1962a)
    Mouse                 M/F   Intraperitoneal   Water              > 1000       Carlson & Highstrete 
      (TUC/ICR)                                                                   (1968)
    Rat (TUC/SPD)         M     Intraperitoneal   Water              > 2000       Carlson & Highstrete 
                                                                                  (1968)
    Rat (TUC/SPD)         M     Subcutaneous      NR                 > 5000       Ray & Ceru (1969)
    Rat (NR)              NR    Oral              Methylcellulose    > 8000       Carlson & Ceru (1965)
    Rat (Sprague-Dawley)  M/F   Oral              Corn oil           > 33         Goyings et al. (1970a)
    Rat (Sprague-Dawley)  M/F   Oral              Propylene          > 22         Goyings et al. (1970b)
                                                  glycol
    Rabbit (albino)       M/F   Skin (abraded)    Corn oil           > 22         Goyings et al. (1970c)
    Rabbit (albino)       M/F   Skin (intact)     Corn oil           > 22         Goyings et al. (1970c)
    Rabbit (albino)       M/F   Skin (abraded)    Propylene          > 22         Goyings et al. (1970d)
                                                  glycol
    Rabbit (albino)       M/F   Skin (intact)     Propylene          > 22         Goyings et al. (1970d)
                                                  glycol
                                                                                                          

    NR, not reported
    

          Mice 

         In a pilot study, which did not conform to GLP, intended as a
    range-finding probe for the minimally effective dose of melengestrol
    acetate for estrus inhibition and for a study of carcinogenicity,
    groups of five adult female ICH mice were given a dose of 0.033,
    0.166, 0.33, 1.3, 3, 5, or 7.5 mg/kg bw per day orally for 10 days.
    There was no untreated control group, and the method of administration
    and the vehicle were not reported. The animals were observed for a
    further 20-23 days for changes in body weight and for the estrus cycle
    by vaginal smears. With large individual variation, the minimally
    effective dose for inhibition of the estrus cycle was 3-5 mg/kg bw per
    day and was calculated to be 4.2 mg/kg bw per day. No
    treatment-related changes in body weight and no deaths were observed
    (Goyings & Kaczkofsky, 1969a).

         In a study that did not conform to the principles of GLP, groups
    of five adult TUC-ICR mice of each sex were treated orally with
    melengestrol acetate by gavage at a dose of 0, 1, 3, 10, or 30 mg/kg
    bw per day for 30 days. The control groups received the vehicle (0.25%
    methylcellulose) only. Clinical signs and body weights were recorded
    daily. At termination, haematocrit, haemoglobin, differential
    leukocyte counts, and gross and histological appearance were
    evaluated. The body weight of mice at 3 mg/kg bw per day was slightly
    increased and that of mice at the highest dose was lower than that of
    controls. There were no clinical signs or haematological changes
    attributable to treatment. Owing to its progestational effect,
    melengestrol acetate depressed the uterine and ovarian weights of mice
    at the high dose, and no corpora lutea were present at doses > 3
    mg/kg bw per day. No gross or microscopic lesions associated with
    treatment were reported. The depression of body weight was presumed to
    be due to the corticosteroid acivity of melengestrol acetate. The NOEL
    was 1 mg/kg bw per day (Goyings & Kaczkofsky, 1969b).

         In a preliminary study, which did not conform to GLP, to
    determine the effects of melengestrol acetate on serum prolactin and
    growth hormone concentrations and on mammary tumour development,
    melengestrol acetate was administered in the diet to groups of five
    puberal female C3Han/f mice, providing doses equal to 0, 0.05, 0.25,
    0.5, 1.5, 2.5, 5, and 25 mg/kg bw per day for 20-21 days. At
    termination, the body weights were recorded, the uteri and ovaries
    were examined histologically, and the serum concentrations of
    prolactin and serum growth hormone were determined by
    radioimmunoassay. No raw data for independent evaluation of the
    conclusions were submitted. Melengestrol acetate induced a
    dose-dependent increase in body weight, which was statistically
    siginificant at doses > 2.5 mg/kg bw per day when compared with
    controls. Dose-related changes in uterine weight were triphasic and
    significantly increased only at the high dose; ovarian weights were
    not affected. The results of histological evaluation of these organs
    were not reported. The serum prolactin concentration at the highest
    dose was statistically significantly higher then that of the control

    and of mice in all other groups. Melengestrol acetate did not alter
    the serum concentration of growth hormone (Lauderdale et al., 1972). 

         Groups of five sexually mature female ICR and CH3Han/f mice were
    fed diets containing melengestrol acetate to provide a dose of 0,
    0.25, 0.5, 2.5, 5, 10, 15, 20, 25, or 40 mg/kg bw per day for 20 days.
    At termination, the animals were necropsied; the mammary glands were
    examined microscopically and scored for development on the basis of
    duct branching. The study did not comply with GLP, the report was
    incomplete, and the copy was partly unreadable. No deaths were
    reported throughout the experiment. In ICR mice, mammary duct
    proliferation was not different from that in controls, but C3Han/f
    mice showed a significant, dose-related increase in mammary duct
    proliferation at doses > 15 mg/kg bw per day when compared with
    controls. Owing to the study design and the high grading of mammary
    gland development in the C3Han/f control group, no NOEL could be
    identified (Charron et al., 1973).

         In a study that did not comply with GLP, weanling female C3Han/f
    mice received melengestrol acetate in the diet for 20 days at a
    concentration of 0, 2.5, 7.5, 12.5, 25, 50, or 125 mg/kg, equivalent
    to doses of 0, 0.5, 1.5, 2.5, 5, 10, and 25 mg/kg bw per day. The
    study was intended to elucidate the effect of melengestrol acetate on
    prolactin serum concentrations and mammary gland development in the
    absence and presence of the prolactin inhibitor
    6-methyl-8-ergoline-acetonitrile (MEA). Clinical signs were monitored
    daily and body weights at the beginning and end of the experiment, but
    the results of these observations were not reported. At termination,
    the serum prolactin concentra-tion was measured, and the mice were
    examined histologically for mammary duct proliferation which was
    scored on a six-point scale. Melengestrol acetate significantly
    ( p < 0.05) increased serum prolactin concentrations and mammary
    gland development at all doses, and the effect was partly inhibited by
    MEA. There was no statistically significant association between serum
    prolactin concentration and mammary gland development. No NOEL could
    be identified (Skinner et al., 1980).

         The finding of a melengestrol acetate-induced increase in serum
    prolactin and its inhibition by MEA were confirmed in an additional
    study with female C3Han/f mice treated with the same doses of
    melengestrol acetate and MEA for 20 days (Lauderdale et al., 1980).

          Rats 

         Groups of five juvenile Wistar rats of each sex were treated with
    melengestrol acetate by oral intubation at a dose of 0, 1, 3, or 10
    mg/kg bw per day for 28 days. The study was not conducted under GLP,
    the design did not comply with current standards for short-term
    studies of toxicity, and incomplete data on individual animals were
    submitted. Clinical signs and body weight were recorded daily and food
    consumption weekly. At termination, all animals were monitored for
    haematological parameters, the weights of the liver, kidney,
    reproductive organs, adrenals, spleen, and thymus, and gross

    appearance. Histopathological examinations of 18 organs and tissues
    from two males and two females from each group were carried out. No
    untoward clinical signs and no deaths were reported. Food consumption
    and the average final body weight were reduced and the food conversion
    ratio increased in all treated rats, but the changes were not
    significant at the lowest dose. Terminal haemograms showed a
    dose-related increase in haematocrit and reduced leukocyte and
    absolute lymphocyte counts at the high dose. In females, the absolute
    and relative weights of the adrenals, uterus, and ovaries were
    significantly reduced at all doses when compared with controls. In
    males, the adrenal weights were decreased at the intermediate and high
    doses, and the weights of the thymus and spleen and the absolute but
    not the relative weights of the liver, kidney, and testis were
    decreased at higher doses. Apart from the atrophy of adrenals and
    female reproductive organs, the only treatment-related gross change
    was a reduction in the size of the accessory sex glands in males at
    the two higher doses. The ovaries of most treated females had no
    corpora lutea. The adrenals showed a reduction in cortical mass and a
    loss of zonal differentiation. A dose-dependent increase in fat was
    observed in sternal bone marrow of females. The results indicate that
    melengestrol acetate was toxic at all doses tested, although the
    authors attributed the effects to the progestational and
    corticosteroid activity of melengestrol acetate. No NOEL could be
    identified (Webster & Frielink, 1962b).

         In a conventional 90-day study of toxicity, groups of 10 male and
    10 female Sprague-Dawley rats received melengestrol acetate in the
    diet at a concentration of 0, 280, 2800, or 5400 g/kg, equivalent to
    0, 0.015, 0.15, and 0.3 mg/kg bw per day. The study was carried out in
    compliance with the principles of GLP. Daily clinical observations
    revealed no treatment-related adverse reactions, and no deaths were
    observed. Body weight and food consumption were monitored weekly and
    were not significantly different in treated and control groups.
    Females at the highest dose showed a slight reduction in weight gain
    and food uptake. Urine was collected on days 83-87. At termination,
    blood was collected from 10 animals of each sex for determination of
    haematological and serum chemistry, and the rats were necropsied. The
    haemograms and urinary analysis showed no relevant effects. Females at
    the intermediate and high doses had increased serum cholesterol
    concentrations and alanine aminotransferase activity, but the latter
    values remained within the normal range. Females also showed
    dose-dependent decreases in the weights of the ovaries, uterus, and
    adrenals, which were significant at the highest dose. The only gross
    observation related to treatment was enlargement of the mammary glands
    of females at all doses. The histopathological changes included
    mammary hyperplasia, moderate papillary endometrial hyperplasia,
    agenesis of corpora lutea, and bone-marrow hypoplasia; these changes
    were significant at the intermediate and high doses and some were also
    observed at the lowest dose. The NOEL was 0.015 mg/kg bw per day,
    although marginal hormonal effects were evident even at this dose
    (Paterson & Hall, 1983).

         In a study certified for compliance with GLP and quality
    assurance, groups of 25 weanling Fischer 344 rats of each sex of the
    F1 generation, which had been exposed to the steroid  in utero in a
    study of reproductive toxicity, were given a diet containing
    melengestrol acetate at 0 or 500 g/kg, equivalent to an average dose
    of 0.055 mg/kg bw per day, for 90 days. The animals were examined
    daily for clinical signs, and body weight and food consumption were
    recorded weekly. A full range of haematological, clinical chemical,
    and urinary tests were conducted before the beginning of the study, on
    days 42-44, and at the end in 10 randomly selected females and males
    from each group. In addition, the serum concentrations of
    progesterone, prolactin, and estrogen were determined by
    radioimmunoassay in the selected females at termination. All females
    were monitored for estrus before terminal sacrifice. At the end of the
    study, gross and microscopic pathological examinations were conducted
    on all animals. No treatment-related deaths and no effects on clinical
    appearance, body-weight gain, or food consumption were observed. The
    haemograms of females showed slight but significant increases in
    haematocrit, erythrocyte count, and haemoglobin concentration. The
    only effect of treatment on serum and urinary parameters was a
    significant increase in occult blood in the urine of females.
    Melengestrol acetate significantly decreased the ovarian and adrenal
    weights in females and testis weight in males. No relevant effect on
    ovarian function was seen, as the hormone concentrations, estrus
    cycling and histological appearance of the ovary were similar to those
    in controls. No other treatment-related gross or histological
    alterations were found. The observed effects were presumed to be
    related to the progestational and corticosteroid activity of
    melengestrol acetate. No NOEL could be identified, but 0.055 mg/kg bw
    per day may have been close to the NOEL (Wood et al., 1983).

          Rabbits

         Groups of four mature albino rabbits of each sex were given
    melengestrol acetate intramuscularly at a dose of 50 mg/animal
    (equivalent to 20 mg/kg bw) every second day for 22 days. Controls
    received the same volume of vehicle. The study was not conducted under
    the principles of GLP. Interim checks were conducted for signs of
    toxicity, body weight, and haematological parameters. At termination
    on day 23, serum chemistry and necropsy for gross and histological
    alterations were performed. All animals had noticeable weight loss
    during the last week, accompanied by diarrhoea, emaciation, and
    increased water intake. The haematological findings consisted of a
    marked reduction in the relative number of lymphocytes which was also
    reflected in a decreased total leukocyte count. This effect persisted
    from the first bleeding on day 11 throughout the study. Impaired
    platelet function was found, as indicated by a depression of clot
    retraction. All four treated males died during the last week due to
    massive pericardial and thoracic bleeding after cardiac puncture for
    blood sampling. Melengestrol acetate caused marked alterations in
    serum chemical parameters, including increased concentrations of
    cholesterol and glucose, elevated activities of aspartate
    aminotransferase, lactate dehydrogenase, and alkaline phosphatase, and

    mild decreases in calcium and phosphorus in lipaemic serum. The
    treatment-related gross observations were an enlarged and discoloured
    liver, muscular atrophy, and reduced adrenal size. The histological
    findings included swollen hepatocytes with glycogen deposits,
    cytoplasmatic vacuolar changes, decreased granulation of the zona
    glomerulosa of the adrenals, and slight renal tubular calcification.
    The adverse effects were presumed to be due to the corticosteroid
    activity of this progestogen. No NOEL could be identified (Goyings &
    Kaczkofwski, 1969c).

          Dogs 

         Groups of two beagle dogs of each sex, aged 1-2 years, were given
    melengestrol acetate in gelatine capsules at a dose of 0, 1, 3, or 10
    mg/kg bw per day for 29 days. The study was not conducted under GLP.
    No deaths were observed, and the only treatment-related clinical
    observation was transient, slight-to-moderate diuresis in animals at
    the two higher doses, associated with urine of decreased specific
    gravity at the highest dose at the end of the study. All treated
    animals showed a slight decrease in body weight and increased food
    consumption. The only finding at intermediate and terminal
    haematological examinations was an increased leukocyte count at the
    highest dose which was due to an excessively high value in one dog.
    Some animals at 3 and 10 mg/kg bw per day showed a slight increase in
    alkaline phosphatase activity in interim and terminal blood samples,
    and a mild increase in alanine aminotransferase activity was seen in
    females at the high dose at termination. A dose-related increase in
    the absolute and relative weights of the liver and a reduction in
    adrenal weights was seen at all doses. Further changes in organ
    weights which were not strictly dose-dependent were increases in the
    weights of the kidney (all doses), pancreas (intermediate and high
    doses), and testis (low and high doses) and reductions in the weights
    of the uterus (high dose), spleen (all doses), and lung (all doses).
    Histopathological alterations seen at all doses included cells with a
    pale cytoplasm that did not stain for fat (suggesting glycogen
    infiltration) in the liver, renal tubular epithelium, and zona
    fasciculata of the adrenals, which also appeared narrow. The bone
    marrow of all dogs at the high dose showed a slight increase in
    immature erythrocytes, which was not reflected in peripheral blood. No
    NOEL could be identifed, since most of the treatment-related effects
    were evident at the lowest dose tested (Clark & Albert, 1962).

          Monkeys 

         In a preliminary range-finding study for the hormonal effect of
    melengestrol acetate, groups of eight sexually mature female rhesus
    monkeys were given an apple injected with an ethanolic solution of
    melengestrol acetate at 0, 0.01, 0.1, 0.5, or 1 mg/animal per day to
    provide doses of 1.5, 15, 75, and 150 g/kg bw per day post-menses for
    one menstruation cycle on days 2-36. The study was not conducted in
    compliance with GLP. The animals were surveyed for clinical signs,
    ovulation, and duration of the menstrual cycle. Serum concentrations
    of luteinizing hormone (LH) and follicle-stimulating hormone (FSH)

    were monitored at the beginning for baseline values and during days
    8-16 when the LH peak normally occurs in the menstrual cycle. Samples
    collected for prolactin determination were not assayed. Gonadal
    steroids (estradiol, estrone, and progesterone) were measured in serum
    every second day during days 6-16 and every fourth day until
    termination. Blood samples for hematology and serum chemistry (eight
    indicators of energy metabolism and liver function) were collected
    before treatment and after ovulation (days 23 and 35). At termination
    of the study, a blood glucose tolerance test was completed for each
    monkey. The day of ovulation was designated as the day of the
    periovulatory LH surge associated with the fall in estrogen. Ovulation
    was confirmed by laparoscopy on day 20 of the menstrual cycle. The
    proportion of monkeys that ovulated decreased significantly during the
    treatment cycle from 88% (control and low dose) to 38, 25, and 12% at
    the three other doses. The menstrual cycle was prolonged to 36 and 38
    days at the two highest doses when compared with that of controls (31
    days) and those at the lower doses (29 days). In all monkeys, the
    patterns of change in LH but not in FSH were consistent with the
    appearance of ovulation and the duration of the menstrual cycle. No
    significant effects on estrogens or progesterone were detected among
    animals in the different treatment groups, at different phases of the
    menstrual cycle, or between ovulating and non-ovulating monkeys. The
    results of clinical chemistry, the glucose tolerance test, and
    haematology were not significantly different among treated and control
    groups or between baseline and terminal samples for each group.
    Changes in the LH surge and suppression of ovulation were the most
    sensitive end-points. The NOEL for suppression of ovulation was 1.5
    g/kg bw per day (Hobson et al., 1976).

         In another preliminary study, groups of six sexually mature
    female cynomolgus monkeys were treated orally via a nasogastric tube
    at a dose of 0 (the vehicle, propylene glycol, only), 2.5, 5, or 10
    g/kg bw per day for one menstrual cycle (up to 35 days). This study
    was not conducted in accordance with GLP. The animals were observed
    daily for clinical signs and menstruation, and a blood sample was
    taken for determination of serum concentrations of gonadotropins (LH
    and FSH), gonadal steroids (estradiol and progesterone), and cortisol
    by a validated radioimmunoassay. One monkeys at the low dose and one
    at the high dose were removed from the study because they developed
    anorexia in the second week. All but two monkeys at the intermediate
    and high doses ovulated during treatment, as verified by the serum
    profiles of estradiol and progesterone and the mid-cycle surge of LH.
    One control, two monkeys at 2.5 g/kg bw per day, one at 5 g/kg bw
    per day, and two at 10 g/kg bw per day had a prolonged follicular
    phase resulting in a longer menstrual cycle than controls
    ( p < 0.06). The mean daily hormone concentra-tions and the time
    course of hormone concentrations evaluated with respect to peak height
    and the integrated area under the curve of concentration-time (AUC)
    showed no relevant effects on FSH or progesterone, whereas the AUC for
    LH was decreased during the luteal phase at the low and intermediate
    doses but not at the high dose. The serum estradiol concentration was
    suppressed in the luteal and follicular phases at the higher doses but
    had reached control concentrations by the time of ovulation. The

    cortisol concentration increased steadily in all groups throughout
    treatment, indicating stress, which is known to interfere with
    reproductive processes in primates. Melengestrol acetate thus exerted
    subtle effects on the menstrual cycle of cynomolgus monkeys, but no
    clear hormonal NOEL could be identified, and the results cannot be
    used to predict responses to similar doses of melengestrol acetate
    administered over several consecutive menstrual cycles (Chenault et
    al., 1990).

         In a study to estimate the hormonal NOEL in non-human primates
    according to guidelines established by the US Food and Drug
    Administration and conducted in compliance with the principles of GLP,
    adult female cynomolgus monkeys (5-11 years old) were treated with
    melengestrol acetate over three consecutive menstrual cycles (up to a
    maximum of 105 days) at an oral dose of 0 (ethanol only), 5, 10, or 25
    g/kg bw per day. Eight animals that had had three consecutive normal
    menstrual cycles before treatment were randomly assigned to each dose.
    Two animals, one at the low and one at the intermediate dose, were not
    included in the final evaluation because they were not cycling
    normally before treatment. Clinical signs and menses were observed
    daily. Blood samples for determination of serum concentrations of
    estradiol, progesterone, LH, FSH, and cortisol by radioimmunoassay
    were collected daily during the last menstrual cycle before treatment
    and the last (third) cycle of the treatment period. Only progesterone
    concentrations were monitored every second day of the first cycle
    after start of treatment and every third day during the following
    cycle. The height of the maximum peak and the time course of hormone
    concentrations in serum were evaluated. The occurrence of ovulation
    was defined by an increase in serum progesterone concentration to
    > 2 ng/ml, and the time of ovulation was determined by the
    periovulatory LH surge, the estradiol peak, and the increase in
    progesterone in the luteal phase. The wide variation among animals in
    the length of the menstrual cycle was minimized by normalizing the
    data for hormones to the ovulatory LH surge. The toxicity of
    melengestrol acetate was not assessed. The hormonal and menstrual
    cycle variables showed the changes expected in response to a
    progestogen, such as significantly decreased serum concentrations of
    LH and estradiol, at the intermediate and high doses, and of
    progesterone, at the highest dose. Significantly fewer animals (three
    of eight) at the highest dose menstruated and ovulated, and changed
    cycles were seen in significantly more animals at the intermediate
    dose (five out of seven) and high dose (five out of eight). The
    dose-related prolongation of the first cycle did not reach statistical
    significance in the other cycling animals. The serum concentrations of
    FSH and cortisol were not affected by melengestrol acetate. Although
    the authors concluded that melengestrol acetate at a dose of 5 g/kg
    bw per day had no effects of biological significance on any of the
    hormonal response parameters, the effects at the lowest dose, even
    though not statistically significant, were consistent with the
    hormonal response seen at higher doses. Therefore, the lowest dose of
    5 g/kg bw per day was the minimally effective dose and was close to
    the NOEL for hormonal effects (Chenault et al., 1993).

          Cattle 

         In studies to evaluate the progestational efficacy of
    melengestrol acetate, five heifers were fed melengestrol acetate in
    their daily ration at a concentration of 0.0625 mg/head, equal to 0.16
    g/kg bw per day, assuming a body weight of 400 kg, for 15-116 days
    after estrus. Melengestrol acetate reduced the number of animals in
    estrus by 40% without inhibiting the formation of corpora lutea, which
    were found in all animals. Concentrations of 0.25 and 0.4 mg/head per
    day, equal to about 0.7 and 1.1 g/kg bw per day, respectively, given
    to groups of 5-18 heifers significantly increased the follicular fluid
    weight and reduced the number of animals with corpora lutea to
    < 6%. No hormonal NOEL could be identified (Zimbelman & Smith,
    1966a,b; Piedkalns, 1971). 

         Melengestrol acetate fed to heifers at a concentration of 0.45
    mg/head per day, equal to 1.8 g/kg bw per day, assuming a mean body
    weight of 250 kg, from 2.5 to 11.3 months of age significantly
    increased the serum concentrations of estradiol-17 and estrone and
    decreased that of progesterone throughout the estrus cycle when
    compared with random values for untreated heifers (Purchas et al.,
    1971a; Lauderdale 1977b). The concentrations of hormones in
    melengestrol-treated animals resembled those during proestrus
    (Echternkamp & Hansel, 1971; Henricks et al., 1971; Weetemann et al.,
    1972). Melengestrol acetate significantly suppressed the serum
    concentration of cortisol to about 50% of the values in control
    animals and that of corticosterone from 1.4 ng/mL in controls to 0.6
    ng/ml (Purchas et al., 1971a,b; Lauderdale, 1977b). This effect was
    attributed to the known negative feedback caused by progestogens on
    the production of the corticotropin-releasing hormone (Manigli et al.,
    1966; Purchas et al., 1971b). The serum concentration of growth
    hormone was not significantly changed (Purchas et al., 1971b). No NOEL
    could be identified for the progestational and corticosteroid activity
    of melengestrol acetate in cattle.

         In a study of reproductive toxicity (see section 2.2.5), 46 cows
    of various breeds received melengestrol acetate in their diet at a
    dose of 0 (26 animals) or about 1 mg/head (equal to about 2 g/kg bw
    per day; 20 animals) for 496-655 days. The melengestrol
    acetate-containing diet was removed only to allow breeding. At
    termination, all animals were necropsied. The adrenal weights were
    reduced in animals fed melengestrol acetate, but gross and
    histological examinations revealed no treatment-related evidence of
    tumour induction or of other abnormalites. No NOEL was identified
    (Goyings, 1971a). 

         In a study of reproductive toxicity (see section 2.2.5), groups
    of bulls of various breeds received melengestrol acetate in the diet
    at a dose of 0 (17 animals) or 1 mg/head, equal to about 2 g/kg bw
    per day (16 animals) for 2 years. Thirty-two days after breeding and
    after a total duration of treatment of 774 days, the animals were
    slaughtered and evaluated for gross and microscopic appearance. The
    adrenal weights were significantly reduced, from 27 g in controls to

    24 g in treated bulls. Several fortuitous lesions were observed. None
    of the gross or microscopic lesions detected could be attributed to
    treatment. No tumours were observed in either group. No NOEL was
    identified (Goyings, 1971b). 

    2.2.3  Long-term studies of toxicity and carcinogenicity

          Mice 

         In a lifespan study of carcinogenicity that did not comply with
    GLP, melengestrol acetate was incorporated into the diet of groups of
    61-71 juvenile ICR mice of an outbred strain of each sex at
    concentrations providing doses equal to 0, 0.017, and 17 mg/kg bw per
    day. The mice were surveyed for 24.5 months. Clinical signs, deaths,
    and body-weight gain were evaluated, and all animals found dead or
    sacrificed at interim and all survivors at termination were autopsied
    for evaluation of gross and microscopic anatomical appearance. Males
    and females at the highest dose were significantly heavier than the
    controls and those at the low dose at all weighings. The survival rate
    of females at the high dose was significantly reduced to 4.4% (from
    21% in controls) at day 746. The reduced survival was attributed to
    the stress of obesity caused by melengestrol acetate. The incidence of
    benign and malignant tumours was unchanged in treated males but was
    decreased in females from 28% in controls to 12% at the low dose and
    18% at the high dose. The mortality rate of females at the high dose
    due to tumours was lower than that of controls. Mammary
    adenocarcinomas were observed in a few females in all groups, with the
    greatest number in the high-dose group: two in controls, one at the
    low dose, four at the high dose. There was no treatment-related
    increase in the incidence of other tumours or of gross or
    histopathological non-neoplastic lesions. Although the authors
    concluded that melengestrol acetate is not carcinogenic under the
    conditions of the study, no firm conclusions could be drawn
    (Lauderdale & Goyings, 1972).

         In a similar lifetime study, which did not comply with GLP,
    melengestrol acetate was fed at the same doses to groups of 64-71
    prepuberal weanling C3Han/f mice of each sex for a maximum of 33
    months (Lauderdale & Goyings, 1972). Previous short-term studies had
    demonstrated that this strain of mice is more sensitive than ICR mice
    to the hormonal effects of high doses of melengestrol acetate on
    mammary gland development (Lauderdale et al., 1972). Melengestrol
    acetate significantly increased the body weight of females at the high
    dose during the first 24 months and reduced their lifespan when
    compared with controls. In males, melengestrol acetate did not affect
    the number of malignant tumours and decreased the incidence of benign
    tumours. In females, the frequency of malignant tumours was reduced at
    the low dose (19 tumours) and significantly increased at the high dose
    (41 tumours) when compared with controls (27 tumours). The higher
    incidence of malignant tumours was the result of an increase in the
    number of mammary adenocarcinomas, from 8 in controls to 10 at the low
    dose and 35 at the highest dose. The frequency of benign tumours was
    slightly decreased in treated females. The only effect on

    non-neoplastic gross and histopathological lesions was hyperplastic
    endometriosis in four females at the high dose, which was not
    statistically significant. Because of the greater susceptibility of
    C3Han/f mice to the hormonal effects of melengestrol acetate, the
    increased rate of mammary tumours was presumed to be due not to a
    direct carcinogenic effect of melengestrol acetate but to a promoting
    effect of elevated prolactin concentrations on mammary gland
    development.

         In another lifespan study, post-puberal female C3Han/f mice aged
    63-84 days, 77-91 days, 84-105 days, 98-112 days, or 119-126 days at
    the beginning of the study were treated with melengestrol acetate
    (Goyings et al., 1976). Previous results (data not submitted) had
    indicated that 44-day-old female C3Han/f mice had higher serum
    prolactin concentrations than those that were 100 days old. The study
    was based on the premise that mice fed melengestrol acetate from an
    early age would be more sensitive to prolactin and have greater
    mammary gland development, providing more potential sites for
    interaction with other factors for mammary tumour formation, such as
    mammary tumour virus. Groups of 16 animals per age group and 80
    animals per dose received a diet containing melengestrol acetate at a
    concentration of 0, 2.5, 5, 7.5, 12.5, 25, 50, 75, or 125 g/kg of
    diet, equal to 0, 0.5, 1, 1.5, 2.5, 5, 10, 15, and 25 mg/kg bw per
    day. The study was terminated after 27 months when the mortality rate
    had reached 90%. The study was not conducted in compliance with GLP. 

         No clinical signs related to treatment were observed, and the
    body weights were similar at the different doses. There was a
    significant effect of age on the incidence of all tumours and of
    mammary tumours, with a higher frequency in the youngest group of both
    treated and control animals. The mean mammary tumour incidence in all
    control animals was 3.8%, which was markedly lower than the incidence
    of 25% observed in a previous study with this mouse strain in which
    the animals were younger (42-44 days) at start (Lauderdale & Goyings,
    1972). No clear dose-response relationship was observed in the number
    of tumour-bearing or mammary tumour-bearing mice, the highest
    incidences being found at 50, then 7.5, 25, 12.5, and 125 mg/kg of
    diet. The incidences in the youngest groups were significantly
    different from those of controls. The higher incidence of all tumours
    was due to the increase in the number of mammary tumours. The reason
    for the low incidence in the group given 75 mg/kg of diet was not
    ascertained. Melengestrol acetate had no effect on the time at which
    tumours were first detected. The other gross and microscopic lesions
    reported were considered to be spontaneous and were observed in all
    groups independently of treatment, except for uterine changes. A
    significant increase in the incidence of cystic endometrial
    hyperplasia with metritis was detected at concentrations > 25 mg/kg
    of diet when compared with controls and was suggested to be a
    progestational effect. This concentration (equal to 5 mg/kg bw per
    day) was stated by the authors to be the minimal effective dose for
    biological effects, but the NOEL for mammary tumours was 1 mg/kg bw
    per day. The finding that younger mice which are more sensitive to
    prolactin have a higher mammary tumour incidence indicates that

    melengestrol acetate is not a directly acting carcinogen but cause
    tumours by releasing prolactin. 

         In a study to investigate the relationship between long-term
    administration of melengestrol acetate, serum prolactin concentration,
    mammary duct proliferation, and mammary tumour development, groups of
    20 adult female C3Han/f mice (44 days of age) were fed melengestrol
    acetate in the diet at a concentration of 0, 2.5, 7.5, 12.5, 25, 50,
    or 125 mg/kg, equivalent to 0, 0.5, 1.5, 2.5, 5, 10, and 25 mg/kg bw
    per day, for 1 year. Additional groups fed 0, 25, 50, and 125 mg/kg of
    diet were also given a daily subcutaneous injection of 100 g/mouse of
    the prolactin inhibitor MEA; however, the dose given (1.9-49 g/mouse)
    was too low to be effective. The study was performed under GLP. Daily
    examinations showed no treatment-related adverse effects. Mean body
    weights, recorded every 2 weeks, were increased in animals at the
    highest dose in the absence and presence of MEA, by 16 and 19%,
    respectively, over those of controls at termination. The rate of
    survival was similar in treated and matched control groups. At the
    time of terminal kill, blood was collected for determination of
    prolactin by radioimmunoassay, and mammary gland development was
    examined histologically and rated on a six-point scale for mammary
    duct branching. Serum prolactin concentrations were increased at all
    doses, with a significant increase at doses > 10 mg/kg bw per day
    of melengestrol acetate alone and at 25 mg/kg bw per day bw of
    melengestrol acetate plus MEA when compared with controls. The hormone
    concentrations of MEA-treated groups were lower than those of the
    corresponding groups that did not receive MEA. A trend of increased
    mammary gland development was observed at a dose of melengestrol
    acetate of 2.5 mg/kg bw per day, which was significant at higher
    doses, in the absence and presence of MEA. The NOEL for hormonal
    effects was close to 0.5 mg/kg bw per day (Raczniak et al., 1981).

         In a follow-up study to test the hypothesis that the increased
    incidence of mammary tumours in mice was due to melengestrol
    acetate-induced enhancement of serum prolactin concentrations, a
    lifetime study was conducted with a similar design with respect to
    animals and doses of melengestrol acetate and MEA (100 g/mouse per
    day), on the premise that the increased formation of mammary tumours
    would be reduced in the presence of the prolactin inhibitor. Groups of
    80 female C3Han/f mice were fed melengestrol acetate-medicated diet
    for a maximum of 883 days, when 90% mortality was reached. They were
    observed daily for adverse reactions and deaths. Body weights and
    palpable masses were recorded twice a week. Complete necropsies for
    gross and histopatho-logical changes were conducted on animals that
    died or were sacrificed  in extremis or at study termination.
    Selected mammary tumour tissues were fixed for transmission electron
    microscopy. The study was certified for compliance with GLP and
    quality assurance. During the first year, weight gain increased
    linearly with the concentration of melengestrol acetate and was
    significantly different from that of controls in mice at 15 mg/kg of
    diet. During the second year, body-weight gain was reduced at all
    doses of melengestrol acetate, and MEA did not significantly affect
    these changes. The rate of survival (when 90% mortality was reached)

    decreased in a linear fashion with increasing concentration of
    melengestrol acetate in both uninhibited and MEA-inhibited animals
    from 30 months (controls and those at 2.5 mg/kg of diet) to 21 months
    (uninhibited group at 125 mg/kg of diet) and 25 months
    (prolactin-inhibited group at 125 mg/kg of diet). A significant
    difference from controls was found at 22 mg/kg of diet. The survival
    time was significantly longer in prolactin-inhibited groups than in
    matched uninhibited groups. Treatment-related non-neoplastic lesions
    were confined to the reproductive tract and included a decrease in
    cystic ovaries (at all doses, significant at 24 mg/kg of diet) and in
    cystic endometrial glands (at all doses, significant at 4 mg/kg of
    diet) and an increase in cystic endometrial hyperplasia (at all doses,
    significant at 7.5-12.5 mg/kg of diet), uterine adenomyosis
    (significant at all doses), and acute metritis (at the highest dose).
    MEA did not prevent these effects. 

         In mammary glands, adenocarcinomas with various degrees of
    differentiation and occasional benign adenomas were identified.
    Melengestrol acetate significantly affected the numbers of mammary
    tumours and of tumour-bearing animals per group. The dose-response
    pattern was one of increased tumour frequency with dose of
    melengestrol acetate, and the effect became statistically
    significantly different from controls at 7.5 mg/kg of diet. MEA
    partially inhibited mammary tumour development and significantly
    reduced the tumour incidence in treated and control animals.
    Examination of mammary tumours from selected animals at each dose and
    from controls by electron microscopy revealed viral particles commonly
    associated with murine mammary tumour virus. Melengestrol prevented
    the development of ovarian tubular adenomas in prolactin-inhibited and
    uninhibited groups, with a dose-related decrease in incidence and an
    effective dose of 25-50 mg/kg of diet. A significant, dose-related
    increase in the incidence of hepatocellular adenomas was seen in both
    uninhibited and prolactin-inihibited groups at doses of melengestrol
    acetate > 25 mg/kg of diet. An increased tumour incidence was
    observed at 7.5 mg/kg of diet, but the dose-response relationship was
    not consistent up to 25 mg/kg. No treatment-related effect could be
    detected on the incidence of hepatocellular hyperplastic nodules or
    hepatocellular carcinoma. The authors concluded that the reduced
    mammary tumour incidence in melengestrol acetate-treated,
    prolactin-inihibted animals supports the hypothesis that melengestrol
    acetate elicits an indirect modulating action on mammary tumorigenesis
    in female C3Han/f mice by stimulating secretion of prolactin. The NOEL
    for mammary tumorigenesis was 0.5 mg/kg bw per day. No NOEL could be
    identified for ovarian and uterine changes. The minimally effective
    dose for increasing the incidence of hepatocellular adenoma was 5
    mg/kg bw per day (Raczniak et al., 1985). 

         The finding of an increased incidence of liver tumours is not
    consistent with the observations in previous studies of melengestrol
    acetate in mice (Goyings et al., 1971, 1976) in which no evidence of
    treatment-related liver tumorigenesis was reported. A genotoxic
    mechanism seems unlikely, since melengestrol acetate was inactive in a
    battery of tests for genotoxicity (see section 2.2.4). Nongenotoxic

    tumorigenesis in rodent liver can occur by various mechanisms,
    including compound-related hormonal activity, peroxisomal
    proliferation, induction of hepatic drug-metabolizing enzymes, and
    hepatotoxicity. Gross and histopathological evaluation revealed no
    evidence of drug-related hepatotoxic effects or hepatocellular
    peroxisomal proliferation in the study of Raczniak et al. (1985). In
    previous short-term and long-term studies of toxicity, no melengestrol
    acetate-dependent pathomorphological hepatic changes were reported in
    mice (Goyings & Kaczkofsky, 1969b; Lauderdale & Goyings, 1972; Goyings
    et al., 1976). However, no conventional long-term study of the
    toxicity of melengestrol acetate in mice was available which included
    measurement of clinical chemical parameters for the assessment of
    hepatotoxic effects. In rats, no evidence was reported for the
    hepatotoxicity of melengestrol acetate (Webster & Frielink, 1962b;
    Paterson & Hall, 1983; Wood et al., 1983), whereas rabbits showed
    changes in serum chemistry and gross and histological liver morphology
    indicating hepatotoxicity at a dose of 50 mg/kg bw per day (Goyings &
    Kaczkofsky, 1969c). In dogs, only slight and in monkeys no
    siginificant changes indicative of hepatotoxicty were observed (Clark
    & Albert, 1962; Goyings, 1973; Hobson et al., 1976; see below). There
    was little evidence in the open literature that steroid hormones can
    initiate hepatic tumorigenesis, although the stimulation of liver
    growth by hormones may have a modulating effect (Schuppler et al.,
    1983). An influence of increased serum prolactin concentrations can be
    excluded because a similar incidence of liver tumours was found in
    prolactin-inhibited and uninhibited mice. No information was available
    on the effect of melengestrol acetate on the activity of the hepatic
    xenobiotic-metabolizing enzyme systems. The occurrence of
    preneoplastic lesions, an important link in nongenotoxic liver
    tumorigenesis, was not recorded. The observed hepatocellular
    hyperplastic nodules, which were not increased in frequency by
    melengestrol acetate, were not further characterized histochemically.
    Other confounding factors are the greater sensitivity of some mouse
    strains to hepatotoxic effects and the variable incidence of
    spontaneous liver tumours. Historical data on the C3Han/f mouse strain
    used in this study were not available. Owing to the lack of
    information, the mechanism of hepatocellular tumorigenesis by
    melengestrol acetate in female C3Han/f mice remains unclear.

          Dogs 

         In a study that did not comply with GLP, melengestrol acetate was
    administered orally to beagles (age not stated) in gelatin capsules at
    a daily dose of 0 (three males and 10 females), 1 g/kg bw (three
    males and 20 females), or 2 g/kg bw (three males and 10 females) for
    2 years and at 8 g/kg bw per day (three males and 10 females) for 1
    year followed by 4 g/kg bw per day for another year. The purpose of
    this study was to investigate the effects of melengestrol acetate at
    doses near those that suppressed estrus in females. No
    treatment-related adverse effects were seen at the low and
    intermediate doses or in males at the high dose, but melengestrol
    acetate suppressed estrus in all females at 8 g/kg bw per day and at
    the subsequent dose of 4 g/kg bw per day. Animals at the high dose

    showed clinical signs related to the progestational activity of
    melengestrol acetate, such as pyometria and dystocia, during the
    second year. Determinations of serum chemical and urinary end-points
    at eight intervals during treatment showed no statistically
    significant abnormalities in males or females at the low and
    intermediate doses, but females at the high dose showed a significant
    increase in serum alkaline phosphatase activity at the last two
    samplings. None of the other changes in blood chemistry or urinary
    end-points showed consistent dose- or time-dependent relationships, or
    they remained within the normal range. Simultaneous haematological
    tests revealed significant effects after 18 months only in females at
    the high dose. These included increased total leukocyte counts due to
    segmented neutrophils and reduced erythrocyte count, haemoglobin, and
    haematocrit. Most of these changes occurred in females with
    melengestrol acetate-induced abnormalities of the reproductive tract.
    At necropsy, the weights of the uterine cervix showed a dose-related
    increase at all doses of melengestrol acetate when compared with
    controls; this effect was considered to be associated with the
    reproductive status of the animals. No other significant
    treatment-related effect on organ weights was observed. The changes
    observed in males were not related to the treatment. Gross and
    micoscopic examination revealed palpable mammary nodules in one female
    each in the control, low-, and intermediate-dose groups.
    Histologically, these nodules appeared to consist of normal
    lobuloalveolar tissue without evidence of malignancy or preneoplastic
    changes. Thus, in females which are sensitive to progestational
    substances, melengestrol acetate induced no neoplastic changes of the
    mammary gland at the highest dose tested. The only treatment-related
    histopathological changes found in females at the high dose were
    alterations of the endometrium which are characteristic of
    progestational agents. When matings were made within the treated
    groups, progestational effects were found to be the most sensitive
    end-points of long-term exposure of females to melengestrol acetate.
    All of the effects were considered to be associated with the hormonal
    activity of melengestrol acetate. The NOEL for hormonal effects was 1
    g/kg bw per day. There was no evidence of carcinogenicity (Goyings,
    1973). 

         In another study, dogs received an intramuscular injection of a
    sustainable release formulation of melengestrol acetate every 3 months
    for 12 months (Carlson, 1968; Carlson & Hall, 1968). Owing to the
    unknown toxicokinetics and systemic exposure of the animals to
    melengestrol acetate in this formulation, the study was not considered
    further.

          Monkeys 

         Female rhesus monkeys received an intramuscular injection of a
    sustainable release formulation of melengestrol acetate every 3 months
    at 6, 20, or 60 mg/kg bw. No control group was available. All doses
    induced hormonal effects (Carlson & Hall, 1968; Carlson, 1969). Owing
    to the unknown toxicokinetics and systemic exposure of the animals to
    melengestrol acetate in this formulation, this study was not
    considered further.

    2.2.4  Genotoxicity

         The results of studies of the genotoxicity of melengestrol
    acetate are summarized in Table 2. The validity of the tests was
    checked with adequate positive control substances of known
    genotoxicity in the respective test systems. Melengestrol acetate,
    which does not contain a stuctural alert, was inactive in a battery of
    tests in prokaryotes and eukaryotes  in vitro and in one system
     in vivo. 

    2.2.5  Reproductive toxicity

         (a)  One-generation studies

          Rats 

         In a study of reproductive toxicity that complied with GLP,
    melengestrol acetate was administered to groups of 15 male and 30
    female Fischer/344 rats in the diet at a concentration of 0, 500,
    1000, 2000, 4000, or 8000 g/kg, equivalent to 0, 0.03, 0.06, 0.13,
    0.25, and 1 mg/kg bw per day. Females were fed medicated diet for 14
    days and males for 60 days before mating, and treatment was continued
    for another 55 days throughout breeding, gestation, lactation, and
    weaning. Daily clinical observations showed no treatment-related
    adverse reactions. No significant effects on body-weight gain before
    breeding were observed. Estrus, monitored by vaginal cytological
    examinations before breeding, was suppressed at doses > 2000 g/kg
    of diet, while at lower doses all animals but one entered the estrus
    cycle. The number of dams was at least 25 in controls and in the group
    at the lowest dose, seven at 1000 g/kg of diet, and none or one at
    higher doses. The fertility and pregnancy rate of dams were slightly
    decreased at 500 g/kg of diet but were not significantly different
    from controls, but only one animal at 1000 g/kg of diet became
    pregnant. In 10 selected inseminated animals from the control group
    and those at 500 g/kg of diet killed on day 13 of gestation, no
    difference in the implantation efficiency (number of implantation
    sites/number of corpora lutea) was observed, while the incidence of
    resorption was doubled in treated dams. The length of gestation was
    not affected by treatment. The litter size, number of live newborns,
    and pup sex ratio showed no drug-related changes. No grossly abnormal
    newborns were recorded. The pup body weights on lactation days 0, 4,
    14, and 21 were not significantly different, although the group given
    500 g/kg of diet had a higher mean pup body weight at all weighings
    when compared with controls. The mortality rate of pups during
    lactation was not affected by treatment. Before terminal sacrifice of
    the F0 animals, blood was taken for haematological and serum
    chemical measurement and for determination of prolactin, progesterone,
    and estrogen concentrations in the serum of females. The haemograms
    showed slight, biologically insignificant variations, such as
    increased haemoglobin and mean corpuscular volume at the highest dose,
    increased nucleated erythrocytes, decreased platelet counts, and
    atypical lymphocytes at all doses. Females were more affected than
    males. Serum chemical values were reported only for males. The only

    change observed was in the activity of aspartate aminotransferase,
    which remained within the normal range. In females, melengestrol
    acetate did not affect the serum estrogen concentration but
    significantly increased the serum prolactin concentration at doses
    > 1000 g/kg of diet and significantly decreased the progesterone
    concentration at doses > 2000 g/kg when compared with controls.
    The weights of the adrenals, ovaries, and uterus showed linear,
    dose-dependent decreases in all treated females, which were
    significantly different from those of controls at 1000 g/kg of diet.
    No treatment-related effects were seen in the weights of the adrenals
    or selected reproductive organs from males. The only remarkable
    observation on gross necropsy was small, dark adrenal glands in
    females at the highest dose. Histological evaluation revealed
    characteristic progestational changes in the ovaries and uteri of
    treated dams, as indicated by the dose-related inihibition of
    ovulation, suppression of corpora lutea development, and increased
    papillary endometrial hyperplasia. These changes were significant at
    doses > 2000 g/kg of diet. Male reproductive organs showed no
    treatment-related morphological changes. The observed effects were
    considered to be associated with the hormonal activity of melengestrol
    acetate. The NOEL for maternal toxicity, male fertility, and litter
    development was 500 g/kg of diet, equivalent to 0.03 mg/kg bw per
    day, a dose that was still hormonally effective (Raczniak et al.,
    1983).

         Further studies on the reproductive toxicity of melengestrol
    acetate in rats receiving a single intramuscular or subcutaneous
    injection of a sustainable release formulation were available
    (Cornette & Duncan, 1968; Bollert et al., 1970a). Owing to the unknown
    toxicokinetics and systemic exposure of the dams to melengestrol
    acetate in this formulation, the studies were not considered further.

          Dogs 

         None of the studies described below conformed to GLP, and they
    fell short of current standards for studies of reproductive toxicity.

         In a range-finding study for dose selection, groups of four
    females of unknown age were treated orally with melengestrol acetate
    at a concentration of 10, 50, 100, 200, 400, or 800 g/animal per day,
    equal to 1, 5, 10, 20, 40, and 80 g/kg bw per day, for periods up to
    240 days or until estrus occurred. No control group was available. The
    animals were observed for estrus and bred at estrus during or after
    treatment to assess the effects of the drug on fertility. Doses
    > 200 g/animal suppressed estrus in all females. Lower doses were
    only partly effective or had no effect on estrus. All animals but one
    cycled during or after treatment, but animals in which cycling had
    been inhibited cycled at progressively longer intervals as the dose
    increased, from 42 days at 100 g/animal to 157 days at 400 g/animal.
    All females that cycled were bred and whelped normal pups. The study
    was inadequate for determining a NOEL for reproductive toxicity
    (Sokolowski & Van Ravenswaay, 1969a).


        Table 2. Results of studies for genotoxicity with melengestrol acetate

                                                                                                                                         
    End-point          Test object        Concentration       S9   Result     GLP    Comments                    Reference
                                                                                                                                         

     In vitro 
    Reverse mutation   S. typhimurium     250-3000 g/plate       Negative   No     Metabolic activation        Zimmer et al. (1978)
                       TA98, TA100,                                                  system not specified
                       TA1537

    Reverse mutation   S. typhimurium     250-2000 g/plate       Negative   Yes                                Mazurek (1982)
                       TA98, TA100,
                       TA1535, TA1537, 
                       TA1538

    Forward mutation   V79 cells (hprt    2.5-10 g/ml            Negative   Yes    Positive at 5 g/ml with     Harbach et al. (1982)
                       locus)                                                        S9 in one experiment;
                                                                                     no significant effect in
                                                                                     second experiment

    DNA damage         Rat primary        0.25-1000 g/ml          Negative   Yes    Cytotoxic at doses          Raczniak (1982)
    (unscheduled       hepatocytes                                                   > 500 g/plate
    DNA synthesis)

    DNA damage         V79 cells          0.03-1.0 mmol/L         Negative   No     Cytotoxic + S9 at           Petzold & Bedell 
    (alkaline elution                                                                1.0 mmol/L; not             (1978)
    assay)                                                                           specified whether 
                                                                                     S9 from mouse or rat 
                                                                                     liver

     In vivo 
    Micronucleus       Mouse bone         250-100 mg/kg bw         Negative   Yes    Two intraperitoneal         Trzos & Swenson 
    formation          marrow                                                        injections separated by     (1982)
                                                                                     24 h
                                                                                                                                         

    S9, 9000  g supernatant of rodent liver; GLP, good laboratory practice
    

         The effects of the minimally effective dose for estrus inhibition
    of 100 g/animal on conception and gestation were determined in six
    females receiving a daily oral dose 5-16 days before estimated
    parturition or from the day after breeding throughout gestation.
    Treatment had no effect on conception, gestation, or parturition,
    although gestation was slightly prolonged when the drug was given
    throughout. All females delivered normally, and the numbers of live
    and stillborn pups were comparable in the two groups. All of the pups
    appeared grossly normal but were heavier (means: males, 314 g;
    females, 293 g when treated on days 5-16 before parturition; males,
    342 g; females, 325 g when treated throughout gestation) than pups
    from untreated control females that whelped contemporaneously (males,
    277 g; females, 286 g). The higher male:female sex ratio in the group
    treated throughout gestation was considered to be fortuitous
    (Sokolowski & Van Ravenswaay, 1969b).

         The effect of melengestrol acetate on the reproductive
    performance of dogs was evaluated as a part of a 2-year tolerance
    study in beagles and reported after 1 year (Sokolowski & Goyings,
    1972) and at termination (Lauderdale, 1973). The results of the
    tolerance study are described in section 2.2.3. Melengestrol acetate
    was administered orally to beagles (age not stated) in gelatin
    capsules at a daily dose of 0 (three males and 10 females), 1 g/kg bw
    (three males and 20 females), or 2 g/kg bw (three males and 10
    females) for 2 years and at 8 g/kg bw per day (three males and 10
    females) for 1 year followed by 4 g/kg bw per day for another year.
    The treatment of females started 120 days after estrus. The males were
    allowed to breed with females in the same dosage group twice during
    the experiment, and males at the highest dose were further mated with
    females at the lowest dose. Indices of fertility such as breeding,
    conception, gestation, parturition, and appearance of the litter were
    evaluated. At interim and at termination, the doses of 4 and 8 g/kg
    bw per day were found to have suppressed estrus, but all animals
    returned to estrus within 12-201 days after cessation of treatment.
    All females at the low and intermediate doses conceived and whelped,
    whereas the average number of pregnancies at 4 and 8 g/kg bw per day
    was reduced due to a combination of a reduced number of conceptions
    and limited breedings. The length of gestation was not affected by
    treatment. In the first year, whelping and litter parameters were not
    altered at doses up to 8 g/kg bw per day. After breeding in the
    second year, whelping was impaired at the high dose, which resulted in
    significantly greater pup loss. None of the pups was abnormal. The
    percent of male pups and the mean birth weight were slightly but not
    significantly decreased at the high dose. The average weaning weight
    was similar in all groups. There was no evidence of adverse effects of
    melengestrol acetate on the fertility of males. The NOEL for
    reproductive toxicity in females was 2 g/kg bw per day. 

          Cattle 

         None of the studies described below complied with GLP or with
    protocols of conventional studies of reproductive toxicity.

         Sixty-three pregnant Holstein heifers (body weights not stated)
    were fed melengestrol acetate in their basal grain ration at a dose of
    0 or 1 mg/head (equal to 2 g/kg bw per day, assuming an average body
    weight of 500 kg) from day 90 of gestation until day 35  post partum 
    (236 days). Melengestrol acetate had no effect on gestation,
    parturition, the number of stillbirths, or the birth weight of calves.
    Gross and microscopic evaluation of eight cows and four calves from
    each group showed no compound-related abnormalities. The findings in
    uteri and ovaries were within normal limits (Goyings et al., 1966).

         In a study of similar design, 134 cows and heifers of various
    breeds received melengestrol acetate in their diet at a dose of 0 (79
    animals) or about 1 mg/head (equal to 2 g/kg bw per day; 55 animals)
    for 889, 736, or 371 days (Lauderdale, 1971a). The treated diets were
    removed to allow breeding, and the animals thus received melengestrol
    acetate for 297-655 days, representing 67-74% of the length of the
    study in the various groups. The animals were the F1 or F2 progeny
    of melengestrol acetate-treated Holstein heifers in a previous study
    (Goyings et al., 1966). Reproductive performance, as determined by
    estrus, conception rate, and pregnancy rate, was not affected by
    treatment, except for a temporarily reduced conception rate at estrus
    after the last feeding of melengestrol acetate. The average weight of
    calves born to melengestrol acetate-fed cows (33 kg) was lower than
    that of controls (35 kg), but the body weights at weaning were
    similar. In 46 animals selected at random from the treated and control
    groups and slaughtered at termination, no treatment-related effects
    were found in either follicular or corpus luteum development.

         The effect of melengestrol acetate on the fertility of male
    cattle of various breeds was evaluated in groups of 21 or 17 bull
    calves given melengestrol acetate in their diet at 0 or 1 mg/head per
    day, respectively, from weaning at approximately 210 days of age up to
    774 days. The breeding phase was started after 655-745 days of
    treatment, when each bull was mated with two cows which were also
    being treated with melengestrol acetate at 1 mg/head per day. A higher
    percentage of treated bulls refused to mount the first cow when
    compared with controls. The conception rates of cows mated to control
    bulls were 74% at the first service and 86% at the second, and those
    of cows mated to treated bulls were 85% and 91%, respectively. The
    pregnancy rates after 43 days of breeding were 85% for cows bred to
    controls and 91% for those bred to treated bulls. The sperm volume,
    total sperm, percent motile sperm, degree of sperm motility, and time
    to obtain an ejaculate of melengestrol acetate-treated bulls were
    comparable to those of controls. At slaughter 32 days after cessation
    of treatment, the mean testis weights were 657 g in 14 treated bulls
    and 668 g in 17 controls. The results indicate no apparent adverse
    effect on male bovine fertility (Lauderdale, 1970).

         (b)  Developmental toxicity

          Rats 

         In a report of a study of developmental toxicity which did not
    comply with the principles of GLP and did not meet the minimal current
    standards for studies of this type, the data submitted were incomplete
    and did not allow independent confirmation of the adequacy of the
    study or of the conclusions. Groups of 10 female Sprague-Dawley rats
    received a subcutaneous injection of melengestrol acetate at a dose of
    2 mg/kg bw per day (vehicle unspecified) on days 9-20 of gestation.
    The animals were killed on day 20. Only eight animals per group became
    pregnant. The body weight of dams, the number of implantation sites
    (resorbed or dead fetuses), weight of litters, number of pups per
    litter, and sex ratio of pups indicated no effect of treatment.
    Viability, the appearance of pups, and the number of corpora lutea
    were not reported. No conclusions about the developmental toxicity of
    melengestrol acetate can be drawn from this study (Clark et al.,
    1963).

         A single subcutaneous dose of a sustainable release formulation
    of melengestrol acetate was administered to groups of 10 pregnant
    TUC/SPD rats on day 6 of gestation at a dose of 15, 25, 50, or 100
    mg/kg bw. Two groups of 10 animals each received the vehicle only. The
    study did not comply with GLP, and the design and report fall short of
    current standards. As the clinical behaviour, body weight changes, and
    feed intake of the dams were not recorded, maternal toxicity could not
    be assessed. The dams were killed on day 20 of gestation. Litter
    weight and average pup weight were reduced at all doses tested and
    were significantly lower at doses > 25 mg/kg bw when compared with
    controls. The number of resorption sites was increased at doses
    > 15 mg/kg bw, the difference being significant at the highest
    dose. A significantly higher percentage of pups in each litter at the
    highest dose had skeletal abnormalities. The larger numbers of pups at
    50 and 100 mg/kg bw with sternal segment variations such as reduced
    size or absence of various sternebrae, underdeveloped supraoptical
    bones, absence of pubic bone ossification, and open incisive foramina
    indicated retarded development. The incidence of visceral
    abnormalities such as persistent umbilical hernia was increased at 25
    mg/kg bw and was most severe at 100 mg/kg bw, whereas at 50 mg/kg bw
    no such abnormalities were recorded. The occurrence of abnormal rib
    curvature in six pups at the highest dose and in one pup at 50 mg/kg
    bw and the occurrence of short tails in two pups at 100 mg/kg bw were
    considered to represent teratogenic responses to melengestrol acetate.
    A linear dose-response relationship for embryotoxicity and
    teratogenicity was thus seen at doses > 25 mg/kg bw. No NOEL could
    be identified owing to lack of information on the toxicokinetics of
    the sustainable release formulation (Bollert & Highstrete, 1969). 

          Rabbits 

         Melengestrol acetate was administered orally to groups of eight
    pregnant Dutch belted rabbits at a concentration of 0, 0.25, 1, 2.5,
    6.25, 12.5, 25, 50, or 100 mg/kg of diet, equivalent to 0, 0.016,
    0.064, 0.16, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/kg bw per day, on days
    6-18 of gestation. A positive control group received a daily
    subcutaneous injection of 10 mg/animal of melengestrol acetate for the
    same period. The study was not conducted in accordance with the
    principles of GLP. The does were observed daily for clinical
    appearance and weekly for food consumption and body-weight gain. The
    fetuses were removed surgically on day 28 of gestation. Reproductive
    and developmental effects were assessed on the basis of pregnancy
    rates, numbers of resorption sites and macerated fetuses, weights of
    litters and fetuses, numbers of total and live fetuses, sex ratio,
    external anomalies, and visceral and skeletal malformations.
    Implantation efficiency was not determined. The body weights of the
    does increased at doses < 2.5 mg/kg of diet but tended to decrease
    at higher doses, with significantly lower means at doses > 25 mg/kg
    of diet when compared with controls. No other indications of maternal
    toxicity were reported. Melengestrol acetate was fetotoxic at doses
    > 12.5 mg/kg of diet, as indicated by large increases in the
    numbers of resorption sites and macerated and dead fetuses. The
    embryonic mortality rate approached 100% at doses > 50 mg/kg of
    diet. The litter size was reduced at 25 mg/kg of diet. The number of
    live fetuses and mean litter and fetal weights were decreased at 6.25
    mg/kg of diet and significantly lower at doses > 12.5 mg/kg of diet
    when compared with controls. Significant teratogenic effects including
    cleft palate, talipes, umbilical hernia, and incomplete skeletal
    ossification were found at doses of 12.5 and 25 mg/kg of diet. The
    fetotoxic and teratogenic effects were attributed to the
    corticosteroidal activity of melengestrol acetate, as corticosteroids
    have been shown to be fetotoxic and teratogenic in laboratory animals
    and particularly in rabbits (Walker, 1967). At 25 mg/kg of diet, the
    male:female ratio was reduced to 36%. The NOEL for developmental
    toxicity was 0.4 mg/kg bw per day (Goyings et al., 1975). 

         In another study that did not comply with GLP, female Dutch
    belted rabbits received a single subcutaneous injection of a
    sustainable release formulation of melengestrol acetate on day 6 after
    artificial insemination. In a first experiment, groups of 16 animals
    were treated with melengestrol acetate at a dose of 0, 25, or 50 mg/kg
    bw. Necropsy of the does on day 28 of gestation showed that only
    three, four, and seven animals in each group were pregnant. All of the
    embryos of treated does died and were resorbed, while in the
    vehicle-treated control group an average of four fetuses and one
    resorption site per litter were found. In a second experiment, groups
    of 20 rabbits received an injection of 0, 5, or 15 mg/kg bw of
    melengestrol acetate. At termination on day 28 of gestation, 12 and 14
    animals were found to be pregnant. At 15 mg/kg bw, only one live but
    undersized fetus was found in the 12 litters examined, and the average
    numbers of resorptions and macerated fetuses per litter were 2.5 and
    1.8, respectively. The morphological age of the dead fetuses was about

    16 days. At 5 mg/kg bw, five live but undersized fetuses were found in
    two of the 14 litters examined, and there were 1.2 resorptions and 2.6
    macerated and dead fetuses per litter with a morphological age of 20
    days. Examination of 62 live and dead fetuses from does given 5 and 15
    mg/kg bw showed cleft palate in 58, exencephaly in 18, bilateral
    agenesis of the lens in 6, irregular brain conformation in 3,
    umbilical hernia and ablepharia in 2, and enlarged livers in 9
    fetuses. Spina bifida was suggested in several fetuses. Melengestrol
    acetate was thus embryocidal at doses > 15 mg/kg bw and fetotoxic
    at 5 mg/kg bw. The embryotoxicity and developmental toxicity were
    presumed to be related to the corticosteroid activity of melengestrol
    acetate. The clinical appearance of the does was not reported. No NOEL
    could be identified. No information was available on the
    toxicokinetics of the sustainable release formulation of melengestrol
    acetate which would allow estimation of the systemic exposure of the
    does during the sensitive period of gestation on days 6-18 (Bollert et
    al., 1970b).

    2.2.6  Special studies: Immunotoxicity

         Melengestrol acetate has significant glucocorticoid activity and
    is approximately as potent as hydrocortisone in inducing granulomas in
    the hamster cheek pouch (Duncan et al., 1964). In humans, melengestrol
    acetate has about 1/40th the activity of dexamethasone in suppressing
    serum cortisol concentrations (Nugent et al., 1975). In studies in
    laboratory animals reviewed by Kountz & Wechter (1977), high doses of
    melengestrol acetate had antiinflammatory and immuno-suppressive
    activities comparable to those of high doses of glucocorticoids such
    as hydrocortisone and methylprednisolone. Thus, a dose of 11 mg/kg bw
    of melengestrol acetate reduced the inflammatory oedema caused by
    injection of croton oil. Melengestrol acetate was more potent than
    medroxyprogesterone in inducing oedema in rat hind paws and in
    alleviating adjuvant-induced arthritis. In an experimental model of
    allergic encephalopathy in rats, melengestrol acetate at a daily dose
    of 4 mg/kg bw delayed the onset of paralysis, and a dose of 16 mg/kg
    completely inhibited it. After three weekly subcutaneous doses of 25
    mg/kg bw, some adverse immunosuppressive effects were observed, such
    as spleen atrophy (25%), involution of the thymus (15%), and decreased
    numbers of peripheral leukocytes. These effects were not observed with
    weekly doses of 5 mg/kg bw, and antibody synthesis was not affected.
    Melengestrol acetate increased skin allograft survival in rabbits
    given 50 mg/week, while survival of renal allografts in dogs was not
    prolonged by doses of 40-360 mg/kg twice daily. In combination with
    antilymphocyte sera, melengestrol acetate induced a dose-related
    increase in the survival of heart allografts in rats at doses of 5-50
    mg/kg bw per day. These results indicate that melengestrol acetate has
    immunosuppressive effects at doses > 5 mg/kg bw per day, which is
    markedly higher than the progestationally active dose. In short-term
    studies of toxicity, evidence of immunosuppression, such as decreased
    leukocyte counts in blood, atrophy of the spleen, and thymic
    involution, have been reported at doses markedly higher than the
    minimally effective progestational dose (see sections 2.2.2 and
    2.2.3). 

         In clinical trials, no adverse effects associated with
    immunosuppression were reported during long-term treatment of patients
    (Liggins, 1963; Segaloff, 1965; Phillips, 1969). The dose of
    melengestrol acetate that does not suppress adrenal responsiveness, 10
    mg/person (equal to 0.166 mg/kg bw), can be presumed to be the NOEL
    for immunosuppressive effects. In female cynomolgus monkeys,
    melengestrol acetate at doses < 25 g/kg bw per day did not
    suppress plasma cortisol concentrations, while 5 g/kg bw per day was
    the minimally effective dose for progestational effects (Chenault et
    al., 1993). In cattle, long-term treatment at 0.2 mg/kg bw per day had
    no apparent adverse effect due to immunosuppression, although the
    serum concentrations of endogenous corticosteroids were suppressed to
    50% of the normal values (Purchas et al., 1971a,b; Lauderdale, 1977b).
    In cows, melengestrol acetate did not significantly alter the ability
    of the uterus to resist infection after infusion of  Escherichia 
     coli (Lauderdale, 1971b). The Committee concluded that melengestrol
    acetate has no relevant immunotoxic effect at minimally effective
    progestational doses.

    2.3  Observations in humans

         The results of open clinical trials indicate reasonably good
    tolerance of large daily doses of melengestrol acetate in humans.
    Melengestrol acetate suppressed plasma cortisol concentrations to
    about 20% of pretreatment values in four men and four women given a
    dose of 20 mg/person, with a potency 1/40th that of dexamethasone. The
    NOEL for suppression of adrenal responsiveness was suggested to be 10
    mg/person (Nugent et al., 1975). Long-term treatment of three women
    with endometrial adenocarcinoma at daily doses of 20-60 mg for
    intervals of 5-21 months caused a remarkable regression of the
    malignancies and exerted no apparent adverse effects on liver
    function, haemoglobin, or blood urea nitrogen concentration (Liggins,
    1963; Phillips, 1969). In an unpublished pilot study in which 37
    patients were treated for various types of cancer with daily doses of
    100-300 mg/person or more for periods of 2-26 weeks, the side-effects
    reported were increased appetite (35% of patients), facial fullness
    (24%), increased blood pressure (14%) and blood urea nitrogen (27%),
    or oedema (16%) (Segaloff, 1965). 

         Limited information was available on the hormonally effective
    doses of melengestrol acetate in women. In regularly ovulating women
    (number of volunteers not stated), melengestrol acetate delayed the
    onset of menses at oral doses of 7.5 and 10 mg/day but not at a dose
    of 5 mg/day, equivalent to 80 g/kg bw. In three volunteers, daily
    doses of 2.5 mg with 0.05 mg of ethinyl estradiol suppressed the
    glandular and vascular development of the endometrium. A single dose
    of 5, 7.5, or 10 mg or five daily doses of 2.5 mg (equal to 42 g/kg
    bw) induced withdrawal bleeding in 11 estrogen-primed amenorrhoeic
    women (Duncan et al., 1964). 

         The contraceptive oral dose of melengestrol acetate for women has
    not been reported but is known for progestogens structurally related
    to melengestrol acetate, such as chlormadinone
    (6-chlor-6-dehydro-17alpha-acetoxyprogesterone), medroxyprogesterone
    acetate (6alpha-methyl-17alpha-acetoxyprogesterone), and megestrol
    acetate (D6,6alpha-methyl-17alpha-acetoxyprogesterone). For
    chlormadinone, a contraceptive dose of 0.5 mg/day has been
    recommended. Minimal anti-estrogenic effects on the physical
    properties of the cervical mucus were observed at 50 g, and the
    maximal effect appeared to occur at 300-400 g, at which endometrial
    changes were not prominent (Martinez-Manautou et al., 1967). The
    contraceptive dose of megestrol acetate has been evaluated as 0.35-0.5
    mg/day, whereas a daily dose of 0.25 mg (equal to 4.2 mg/kg bw) had
    little effective (Avenando et al., 1970; Casavilla et al.,1970).
    Megestrol acetate is reported to be more potent in changing the
    cervical mucus than melengestrol acetate (Petrow, 1967). In a
    calculation of the relative potency of the two progestogens based on
    inhibition of menses in estrogen-primed women, melengestrol acetate
    was about 0.72 as potent as megestrol acetate. Thus, 5.8 mg/kg bw of
    melengestrol acetate is presumed to be the minimally effective dose
    for changing the cervical mucus in women. The median effectve dose of
    megestrol acetate for menses inhibition was reported to be > 10 mg
    (Sywer & Little, 1962). 

         An oral dose of medroxyprogesterone acetate of about 1 mg/day is
    the minimally effective dose, as observed from the occurrence of
    withdrawal bleeding (European Medicines Evaluation Agency, 1996). This
    compound is used for contraception as a sustainable release
    formulation at a single intramuscular dose of 150 mg every 3 months,
    equal to 28 g/kg bw per day (Liskin et al., 1987). Data in humans and
    laboratory animals indicate that melengestrol acetate is at least four
    times moreprogestogenic than  medroxyprogesterone acetate. In
    ovulating women, melengestrol acetate at 7.5 mg/day effectively
    delayed menses, whereas medroxyprogesterone acetate was reported to be
    ineffective at a daily dose of 30 mg (Greenblatt & Rose, 1962; Duncan
    et al., 1964). In both the Clauberg-McPhail test in rabbits, in which
    the degree of endometrial proliferation is used as a measure of
    progestational activity, and the pregnancy maintenance assay in rats,
    melengestrol acetate was about four times more potent than
    medroxyprogesterone acetate. The oral NOEL of medroxyprogesterone
    acetate in the Clauberg-McPhail test was 0.03 mg/kg bw per day
    (European Medicines Evaluation Agency, 1996). On the basis of these
    observations, the contraceptive dose of melengestrol acetate in women
    can be assumed to be about 7 g/kg bw per day. 

         The safety of the widely used injectable depot formulation of
    medroxyprogesterone acetate at contraceptive doses has been the
    subject of several multicentre epidemiological studies, many of which
    were conducted under the auspices of WHO (Liskin et al., 1987; Richard
    & Lasagna, 1987). The general conclusions derived from these studies
    were that long-term use of medroxyprogesterone acetate: 

    *    does not increase the overall risks for cancers of the breast,
         cervix, ovary, or liver; 

    *    protects against endometrial hyperplasia and endometrial
         carcinoma in estrogen-treated postmenopausal women; 

    *    does not increase the risk for thromboembolism or other
         circulatory system disease; 

    *    does not impair the function of the adrenal gland; 

    *    has no apparent impact on the immune system; 

    *    causes no clinically important changes in liver function; and

    *    has no harmful effects on fertility, pregnancy, or lactation in
         treated women or on the nursing and development of their
         children.

    3.  COMMENTS

         The Committee considered data from studies of the
    pharmacokinetics, biotransformation, acute toxicity, short-term and
    long-term toxicity, carcinogenicity, genotoxicity, and reproductive
    and developmental toxicity of melengestrol and from studies in humans.
    Most of the studies were conducted before 1979 according to the
    standards in existence at that time and were not carried out in
    compliance with GLP. More recent studies were conducted according to
    the appropriate standards for study protocol and conduct. 

         The results of limited studies on the pharmacokinetics of
    melengestrol acetate in rabbits and humans have been reported. The
    bioavailability of melengestrol acetate after oral administration and
    its kinetics in plasma have not been determined. In studies in which
    radiolabelled melengestrol acetate was used, 3H or 14C was
    inserted at the 6-methyl position. In rabbits, 59% of an orally
    administered dose of [14C]melengestrol acetate was excreted within 7
    days in urine and faeces at a ratio of about 1:3, with a peak
    elimination rate on the first day. In women, the excretion of
    [14C]melengestrol acetate was complete within 10 days, and 74% of
    the radiolabel was recovered in urine and faeces. The half-time
    estimated from the data on excretion was 3-5 days. 

         Limited information was available on the biotransformation of
    melengestrol acetate in cattle, rabbits, and humans  in vivo or in
    cattle and rat liver microsomes  in vitro. Melengestrol acetate is
    extensively metabolized, with the formation of numerous metabolites
    which have been neither adequately identified nor characterized with
    respect to their biological activity. In cattle, intact melengestrol
    acetate accounted for up to 86% of the total radiolabel in fat and for
    29% in liver. In cattle and rat liver microsomes, several mono- and
    dihydroxylated metabolites were identified. In the urine of rabbits,
    two-thirds of the radiolabel was found as glucuronides. The

    6-methyl-hydroxylated metabolite was identified in the free and
    conjugated forms as one of the major metabolites. In humans, 68% of
    the radiolabel in urine was associated with conjugates, whereas faeces
    contained more unconjugated compounds. Peaks representing 13
    metabolites with an intact steroid nucleus were detected. One
    metabolite was identified as 2alpha-monohydroxylated melengestrol
    acetate.

         Melengestrol acetate has little toxicity after a single dose,
    although the studies of acute toxicity were limited as a large volume
    of the vehicle had to be administered. The LD50 values after
    intraperitoneal injection were > 2500 mg/kg bw in mice and > 2000
    mg/kg bw in rats. No deaths were observed among rats given doses of
    8000 mg/kg bw orally or 5000 mg/kg bw subcutaneously. Dermal
    application to the intact or abraded skin of rabbits at the maximum
    achievable dose of 22 mg/kg bw caused no toxic reaction.

         Short-term tests of the toxicity of melengestrol acetate have
    been performed in mice, rats, rabbits, dogs, and monkeys. Melengestrol
    acetate had a greater effect in females than in males, with hormonal
    (progestational and corticosteroidal) effects as the most sensitive
    end-points. 

         In TUC/ICR mice of each sex that received melengestrol acetate
    orally at a dose of 0, 1, 3, 10, or 30 mg/kg bw per day for 30 days,
    the body weights were slightly increased at 3 mg/kg bw per day but
    were decreased at higher doses. Changes in female reproductive organs,
    such as decreased weights of ovaries and uteri relative to body weight
    and the absence of corpora lutea at doses of 3 mg/kg bw per day and
    above were considered to be progestational changes. The NOEL for
    hormonal effects was 1 mg/kg bw per day.

         In a 21-day study, puberal female C3Han/f mice received
    melengestrol acetate in the diet at concentrations providing doses
    equal to 0, 0.05, 0.25, 0.5, 1.5, 2.5, 5, or 25 mg/kg bw per day. Body
    weight was significantly increased at doses of 2.5 mg/kg bw per day
    and above, and the serum concentration of prolactin and the uterine
    but not the ovarian weight were increased at the highest dose. The
    NOEL was 1.5 mg/kg bw per day.

         In mature female ICR and C3Han/f mice given an oral dose of 0,
    0.25, 0.5, 2.5, 5, 10, 15, 20, 25, or 40 mg/kg bw per day for 20 days,
    melengestrol acetate had no effect on mammary duct proliferation in
    ICR mice, but caused a significant, dose-related increase in mammary
    duct proliferation, as indicated by branching of the ducts of the
    mammary gland, in C3Han/f mice at doses of 15 mg/kg bw per day and
    higher. 

         In order to elucidate the contribution of increased serum
    prolactin concentration to melengestrol acetate-induced mammary duct
    proliferation, groups of weanling female C3Han/f mice were given diets
    containing melengestrol acetate at concentrations providing doses of
    0, 0.5, 1.5, 2.5, 5, 10, or 25 mg/kg bw per day for 20 days with or

    without the prolactin inhibitor MEA. The serum prolactin concentration
    and mammary duct proliferation were enhanced at all doses, and the
    effects were partially inihibited by MEA. There was no statistically
    significant association between mammary duct proliferation and serum
    prolactin concentration. A NOEL could not be identified.

         In juvenile rats given melengestrol acetate for 28 days by gavage
    at a dose of 0, 1, 3, or 10 mg/kg bw per day, food consumption and
    body weight were reduced at all doses. Haematological changes were
    also seen, which included a dose-related increase in the erythrocyte
    volume fraction and a decreased leukocyte count in animals at the
    highest dose. In females, the weights of the adrenals, uterus, and
    ovaries were reduced at all doses, associated with atrophy of these
    organs and the absence of corpora lutea in most animals. In males,
    atrophy of the adrenal and accessory sex glands was observed only at 3
    and 10 mg/kg bw per day. The effects reported are consistent with
    progestational and corticosteroidal activity. A NOEL could not be
    identified.

         In a 90-day study of toxicity, rats received melengestrol acetate
    in their diet at concentrations providing doses of 0, 0.015, 0.15, or
    0.3 mg/kg bw per day. The serum cholesterol concentration was
    increased in females at the two higher doses. Changes characteristic
    of the hormonal effects of melengestrol acetate were observed, such as
    decreased weights of the adrenals, ovaries, and uterus at 0.3 mg/kg bw
    per day; mammary gland and endometrial hyperplasia, agenesis of the
    corpora lutea, and bone-marrow hypoplasia at 0.15 and 0.3 mg/kg bw per
    day; and enlarged mammary glands at 0.015 mg/kg bw per day. Other
    effects at the lowest dose, although not statistically significant,
    were consistent with the changes seen at higher doses. The Committee
    concluded that 0.015 mg/kg bw per day was a minimally effective dose. 

         In another 90-day study, weanling rats were fed diets containing
    melengestrol at 0 or 0.055 mg/kg bw per day. The treatment-related
    effects were slight increases in erythrocyte volume fraction,
    erythrocyte count, and haemoglobin concentration and significantly
    lower adrenal, ovarian, and testicular weights. A NOEL could not be
    identified.

         Rabbits were injected intramuscularly with melengestrol acetate
    at 20 mg/kg bw every second day for 22 days. All animals lost weight
    and had diarrhoea. Haematological evaluation revealed decreased
    leukocyte counts and impaired platelet function. All four males died
    during the last week of treatment from thoracic bleeding after blood
    sampling. At termination, serum cholesterol concentrations and the
    activities of aspartate aminotransferase, lactate dehydrogenase, and
    alkaline phosphatase were increased in the surviving females. At
    necropsy, the females were found to have muscular atrophy, reduced
    adrenal size, and enlarged livers with swollen hepatocytes containing
    glycogen deposits. 

         Groups of two male and two female beagle dogs were given
    melengestrol acetate in gelatin capsules orally at a dose of 0, 1, 3,
    or 10 mg/kg bw per day for 29 days. Treatment at 3 and 10 mg/kg bw per
    day induced slight-to-moderate diuresis, with urine of decreased
    specific gravity. Body weight was slightly decreased and food
    consumption increased in all treated animals. Small increases were
    observed in the activity of serum alkaline phosphatase at 3 and 10
    mg/kg bw per day and of serum alanine aminotransferase at 10 mg/kg bw
    per day. A dose-related decrease in adrenal weight and an increase in
    liver weight were seen over the dose range, with histopathological
    changes indicative of glycogen deposition. A NOEL was not identified.

         Groups of eight adult female rhesus monkeys were treated orally
    with melengestrol acetate at a dose of 0, 1.5, 15, 75, or 150 g/kg bw
    per day for one menstrual cycle. Ovulation was monitored by measuring
    the surge of LH and the decrease in estrogen concentration and
    confirmed by laparoscopy. The number of monkeys that ovulated
    decreased significantly during treatment, from 88% in controls and at
    the lowest dose, to 38, 25, and 12% at the three other doses. The
    menstrual cycle was prolonged at the two higher doses, but
    melengestrol acetate had no significant effect on the serum
    concentrations of progesterone and estrogens (estradiol-17 and
    estrone). Changes in the surge of LH and the suppression of ovulation
    were the most sensitive end-points in this study. The NOEL for
    suppression of ovulation was 1.5 g/kg bw per day.

         In a range-finding study for hormonal effects, groups of six
    female cynomolgus monkeys were treated orally with melengestrol
    acetate by nasogastric intubation at a dose of 0, 2.5, 5, or 10 g/kg
    bw per day for one menstrual cycle. One monkey at the lowest dose and
    one at the highest dose were withdrawn from the study because they
    showed anorexia. One monkey at 5 g/kg bw per day and one at 10 g/kg
    bw per day failed to ovulate during treatment. Monkeys at 2.5 and 10
    g/kg bw per day had prolonged menstrual cycles. No consistent
    dose-response relationship was seen for effects on hormone
    concentrations. The serum concentration of estradiol was decreased
    during the luteal phase of the menstrual cycle in animals at 5 and 10
    g/kg bw per day, and luteinizing hormone was suppressed at 2,5 and 5
    g/kg bw per day. Melengestrol acetate had no consistent effect on the
    serum concentrations of progesterone or FSH. The authors concluded
    that 'melengestrol acetate may have exerted subtle effects on the
    menstrual cycle of cynomolgus monkeys'.

         In a follow-up study, female cynomolgus monkeys were given
    melengestrol acetate at a dose of 0, 5, 10, or 25 g/kg bw per day for
    three consecutive menstrual cycles up to a maximum of 105 days. Groups
    of eight animals were observed for three consecutive menstrual cycles
    before treatment. Two animals, one at 5 g/kg bw per day and one at 10
    g/kg bw per day, were not included in the final evaluation because
    their cycles were not normal before treatment. The occurrence of
    ovulation was determined by observing the periovulatory surge of LH,
    the peak of estradiol, and the increase in progesterone concentration
    in the luteal phase. The hormonal and menstrual cycle variables showed

    the changes that would be expected to be induced by a progestogen,
    such as significantly decreased serum concentrations of LH and
    estradiol at 10 and 25 g/kg bw per day and of progesterone at 25
    g/kg bw per day. Significantly fewer animals at the highest dose
    menstruated and ovulated, and significantly more animals at 10 and 25
    g/kg bw per day had changed cycles. In the remaining animals, the
    dose-related prolongation of the first cycle did not reach statistical
    significance. The serum concentrations of FSH and cortisol were not
    affected by melengestrol acetate. The effects at 5 g/kg bw per day,
    although not statistically significant, were consistent with the
    hormonal response seen at higher doses. The Committee considered that
    5 g/kg bw per day was a minimally effective dose and was close to the
    NOEL for hormonal effects.

         In heifers fed melengestrol acetate at a dose equal to 0.16 g/kg
    bw per day for 15-116 days after estrus, treatment reduced the number
    of animals in estrus by 40%, and doses equal to 0.7 and 1.1 g/kg bw
    per day consistently suppressed estrus in all animals. Melengestrol
    acetate was also fed to heifers at a dose equal to 1.8 g/kg bw per
    day from 2.5 through 11.3 months of age. When the animals reached
    maturity, the serum concentrations of estradiol-17 and estrone were
    significantly increased over those in controls, and that of
    progesterone was suppressed to values similar to those occurring in
    proestrus. The serum concentrations of cortisol and corticosterone
    were depressed to about 50% of the concentrations in untreated
    animals. A NOEL could not be identified for the progestational and
    corticosteroid activity of melengestrol acetate in cattle.

         In a study of carcinogenicity, ICR mice received diets containing
    melengestrol acetate at concentrations providing a dose of 0, 0.017,
    or 17 mg/kg bw per day for up to 24.5 months. The animals at the high
    dose weighed more than controls throughout the study, and their
    survival rate was significantly lower. These effects were attributed
    to the stress of obesity caused by melengestrol acetate. The incidence
    of benign and malignant tumours was reduced in treated females but not
    males. A slight, nonsignificant increase in the incidence of mammary
    adenocarcinomas was observed in animals at the high dose. No firm
    conclusion could be drawn about the carcinogenic potential of
    melengestrol acetate in ICR mice.

         In a similar study, prepuberal C3Han/f mice, which were
    previously shown to be more sensitive than ICR mice to the effects of
    melengestrol acetate on mammary duct proliferation, were given diets
    containing melengestrol acetate at concentrations providing a dose of
    0, 0.017, or 17 mg/kg bw per day for up to 33 months. The incidence of
    malignant tumours was increased in females at the high dose, primarily
    because of a large number of mammary adenocarcinomas. This increase
    was assumed to be due not to a direct carcinogenic effect of
    melengestrol acetate but to the promoting effect of increased
    prolactin concentrations.

         In another study, five groups of mature C3Han/f mice aged 63-84,
    77-91, 84-105, 98-112, and 119-126 days were used to assess the effect
    of age on the development of melengestrol acetate-induced mammary
    tumours. The animals received a diet containing melengestrol acetate
    at concentrations providing doses equivalent to 0, 0.5, 1, 1.5, 2.5,
    5, 10, 15, or 25 mg/kg bw per day. The study was terminated after 27
    months, when the mortality rate reached 90%. Age had a significant
    effect on the development of mammary tumours, in both the treated and
    control mice, with the greatest incidence in the youngest group.
    Except for a lower mammary tumour incidence at 10 mg/kg bw per day,
    the incidence increased in a dose-related manner from 1.5 mg/kg bw per
    day. Melengestrol acetate had no effect on the time at which tumours
    were first detected. The treatment-related non-neoplastic lesions that
    were observed consisted of progestational effects, such as increased
    cystic endometrial hyperplasia at doses of 5 mg/kg bw per day and
    greater. On the basis of the finding of a higher incidence of mammary
    tumours in younger animals, which are more sensitive to prolactin, it
    has been postulated that melengestrol acetate is not a directly acting
    carcinogen in C3Han/f mice but cause tumours by increasing the release
    of prolactin. The NOEL for induction of mammary tumours was 1 mg/kg bw
    per day.

         In a study to investigate the relationship between long-term
    administration of melengestrol acetate, serum prolactin concentration,
    and mammary duct proliferation, female C3Han/f mice of 44 days of age
    were fed melengestrol acetate in the diet at concentrations providing
    doses equivalent to 0, 0.5, 1.5, 2.5, 5, 10, or 25 mg/kg bw per day
    for 1 year. Additional groups were also given a daily subcutaneous
    injection of the prolactin inhibitor MEA, but the dose of this
    compound was too low and these groups were not further evaluated. Body
    weights were increased at the highest dose of melengestrol acetate.
    The serum prolactin concentration, which was determined only at
    termination of the study, was increased at all doses of melengestrol
    acetate tested, with a significant increase at doses of 10 mg/kg bw
    per day and higher. An increasing trend in the incidence of animals
    with exacerbated mammary duct proliferation was observed at doses of
    2.5 mg/kg bw per day and above, and significantly increased incidences
    were seen at doses of 5 mg/kg bw per day and more. The NOEL for
    hormonal effects was close to 0.5 mg/kg bw per day.

         In a follow-up study, female C3Han/f mice of 44 days of age were
    fed diets containing melengestrol acetate at concentrations providing
    doses of 0, 0.5, 1.5, 2.5, 5, 10, or 25 mg/kg bw per day, for a
    maximum of about 29 months. Additional groups of animals given 0, 5,
    10, and 25 mg/kg bw per day were also given a daily subcutaneous
    injection of 100 g of MEA. Mice at all doses showed more rapid weight
    gain during the first year but decreased body-weight gain during the
    second year. MEA did not significantly affect the melengestrol
    acetate-induced changes in body weight. The survival rate decreased
    with increasing dose of melengestrol acetate, attaining significance
    at doses of 5 mg/kg bw per day and higher. Mice in which prolactin was
    inhibited survived significantly longer than those in matched groups
    without prolactin inhibition. The only treatment-related

    non-neoplastic lesions observed were decreased numbers of cystic
    ovaries and cystic endometrial glands and increased incidences of
    endometrial hyperplasia, uterine adenomyosis, and acute metritis (at
    the highest dose). MEA did not prevent these effects. In the mammary
    glands of treated mice, adenocarcinomas and occasional benign adenomas
    were identified; a dose-related increase in the incidence of mammary
    tumours was observed, and the incidence of adenocarcinomas in animals
    at doses of 1.5 mg/kg bw per day and higher was statistically
    significantly increased over that in controls. MEA partially inhibited
    mammary tumour development in both control and melengestrol
    acetate-treated groups. Examination of the mammary tumours from
    selected animals at each dose and from controls by electron microscopy
    revealed viral particles commonly associated with the murine mammary
    tumour virus. Melengestrol acetate decreased the incidence of ovarian
    tubular adenomas in animals at doses of 5 mg/kg bw per day and above.
    The incidence of hepatocellular adenomas was signficantly increased in
    animals at doses of 5 mg/kg bw per day and higher, whether or not they
    were treated with MEA, but the dose-response relationship was not
    consistent up to this dose. There was no treatment-related effect on
    the incidence of hepatocellular hyperplastic nodules or hepatocellular
    carcinoma. The Committee concluded that melengestrol acetate
    indirectly modulates mammary tumorigenesis in female C3Han/f mice,
    possibly by stimulating the secretion of prolactin. The NOEL for
    mammary tumorigenesis was 0.5 mg/kg bw per day. A NOEL could not be
    identified for the hormonal effects of melengestrol acetate on the
    ovaries and uterus. The minimally effective dose for increasing the
    incidence of hepatocellular adenoma was 5 mg/kg bw per day. 

         Melengestrol acetate was administered orally to male and female
    beagle dogs at a dose of 0, 1, or 2 g/kg bw per day for 2 years or at
    8 g/kg bw per day for 1 year followed by 4 g/kg bw per day for
    another year. Animals treated at the highest dose showed clinical
    signs of the progestational activity of melengestrol acetate, such as
    pyometria and dystocia, during the second year. Females at the highest
    dose had increased serum alkaline phosphatase activity and, after 18
    months, an increased total leukocyte count and reduced erythrocyte
    count, haemoglobin concentration, and erythrocyte volume fraction.
    Most of these changes occurred in females with abnormalities of the
    reproductive tract. No neoplastic changes were seen in the mammary
    gland at any dose. Females at the highest dose had alterations of the
    endometrium characteristic of progestational activity. Progestational
    effects were the most sensitive end-point. The NOEL for hormonal
    effects was 1 g/kg bw per day. 

         Melengestrol acetate has been tested for genotoxicity in a range
    of assays  in vitro and  in vivo. Gene mutation was not induced in
     Salmonella typhimurium or mammalian cells. Unscheduled DNA synthesis
    was not observed in rat primary hepatocytes or in the alakaline
    elution assay in Chinese hamster V79 cells. Melengestrol acetate did
    not induce micronuclei in the bone marrow of mice exposed  in vivo by
    intraperitoneal injection. The Committee concluded that melengestrol
    acetate is not genotoxic.

         In a one-generation study of reproductive toxicity in rats,
    melengestrol acetate was administered in the diet to provide doses
    equivalent to 0, 0.03, 0.06, 0.13, 0.25, or 1 mg/kg bw per day.
    Melengestrol acetate suppressed the estrus cycle at doses of 0.13
    mg/kg bw per day and above and had significant effects on fertility
    and pregnancy at doses of 0.06 mg/kg bw per day and above: at 0.06
    mg/kg bw per day, only one dam became pregnant, whereas at 0.03 mg/kg
    bw per day all dams became pregnant. While the incidence of resorption
    was double that in controls, there was no difference in litter size.
    The body weights of pups during lactation were not statistically
    significantly different from those of controls, although the birth
    weights of pups of treated dams were higher. Dams at doses of 0.06
    mg/kg bw per day and higher showed significant changes in the serum
    concentrations of estrogen, prolactin, and progesterone and in the
    weights of the adrenals, ovaries, and uterus. The histological
    appearance of the ovaries and uterus was consistent with
    progestational activity. The NOEL for reproductive toxicity was 0.03
    mg/kg bw per day. 

         The effect of melengestrol acetate on reproductive performance in
    beagle dogs was evaluated in the 2-year study described above in which
    melengestrol acetate was administered orally at doses of 0, 1, or 2
    g/kg bw per day, or (to males only) at 8 g/kg bw per day for 2
    years, or (to females only) at 8 g/kg bw per day for 1 year followed
    by 4 g/kg bw per day for another year. Animals treated at the same
    dose were bred, and females at the lowest dose were also bred with
    males at the highest dose. Treatment of females was begun 120 days
    after estrus. Melengestrol acetate at 4 or 8 g/kg bw per day
    suppressed estrus, but estrus resumed within 12-201 days after
    cessation of treatment. Fewer females at the highest dose became
    pregnant, and parturition was impaired in animals at this dose during
    the second year, resulting in significantly greater pup loss. The
    percentage of male pups and the mean birth weight were slightly
    decreased at the highest dose. Melengestrol acetate did not appear to
    affect the fertility of male dogs. The NOEL for reproductive
    performance was 2 g/kg bw per day. 

         Cows and heifers that were F1 or F2 progeny of the
    melengestrol acetate-treated heifers described above received
    melengestrol acetate in their diet at a dose equal to 2 g/kg bw per
    day for up to about 2 years, except during the breeding period.
    Melengestrol acetate completely suppressed estrus. The conception and
    pregnancy rates were not different from those of controls, except for
    a temporarily reduced conception rate at estrus after the last feeding
    of melengestrol acetate. The calves weighed less than those of
    controls at birth but not at weaning. At necropsy, the only
    treatment-related change was reduced adrenal weight. When bull calves
    received the same treatment for about 2 years, no adverse effect was
    seen on fertility, and the only effect was a reduction in adrenal
    weight.

         In a study of developmental toxicity, pregnant rats received a
    single subcutaneous dose of 0, 15, 25, 50, or 100 mg/kg bw of a
    sustained-release formulation of melengestrol acetate on day 6 of
    gestation and were killed on day 20. Reduced litter weights and
    average pup weights, increased numbers of resorption sites, a larger
    percentage of pups with retarded development, and skeletal and
    visceral abnormalities were observed at doses of 25 mg/kg bw and
    higher, and melengestrol acetate was considered to be embryotoxic and
    teratogenic at these doses. A NOEL could not be identified owing to
    lack of information on the toxicokinetics of the sustained-release
    formulation. 

         In a study of developmental toxicity, melengestrol acetate was
    administered orally to pregnant rabbits at concentrations equivalent
    to 0, 0.016, 0.064, 0.16, 0.4, 0.8, 1.6, 3.2, or 6.4 mg/kg bw per day
    on days 6-18 of gestation The fetuses were removed surgically on day
    28. The body weights of the does at doses up to 0.16 mg/kg obw per day
    were increased, and those of animals at higher doses were slightly
    decreased. Melengestrol acetate was embryotoxic and fetotoxic at doses
    of 0.8 mg/kg bw per day and higher, as indicated by a large increase
    in the numbers of resorption sites and dead fetuses. The percentage of
    embryonic deaths approached 100% at the dose of 3.2 mg/kg bw per day.
    The litter size was reduced at 1.6 mg/kg bw per day. The number of
    live fetuses and the mean litter and fetal weights were significantly
    lower at doses of 0.8 mg/kg bw per day and above. The significant
    effects observed at 0.8 and 1.6 mg/kg bw per day included cleft
    palate, talipes, umbilical hernia, and incomplete skeletal
    ossification. At 1.6 mg/kg bw per day, the male:female ratio was
    reduced to 0.36. The Committee concluded that the fetotoxic and
    teratogenic effects of melengestrol acetate in rabbits are due to its
    corticosteroid activity. The NOEL for embryotoxicity and
    teratogenicity was 0.4 mg/kg bw per day. 

         In a study of developmental effects, female rabbits received a
    subcutaneous injection of a sustained-release formulation of
    melengestrol acetate at a dose of 0, 5, or 15 mg/kg bw on day 6 after
    artificial insemination. The fetuses were delivered surgically on day
    28 of gestation. At the highest dose, only one live but undersized
    fetus was found in the 12 litters examined. At 5 mg/kg bw, five live,
    undersized fetuses were found in the 14 litters examined. Nearly all
    live and dead fetuses of treated does had cleft palate. The other
    abnormalities observed were exencephaly, agenesis of the lens,
    irregular brain conformation, umbilical hernia, ablepharia, and
    enlarged liver. Melengestrol acetate was thus teratogenic and
    embryotoxic at a dose of 15 mg/kg bw and fetotoxic at doses of 5 mg/kg
    bw and above. The study is not appropriate for identifying a NOEL. 

         Observations in regularly ovulating women (number not stated)
    indicated that melengestrol acetate delayed the onset of menses at
    doses of 7.5 and 10 mg but not at 5 mg per woman per day. In three
    volunteers, daily doses of 2.5 mg of melengestrol acetate and 0.05 mg
    of ethinylestradiol suppressed endometrial proliferation. Single doses

    of melengestrol acetate of 5, 7.5, or 10 mg or repeated daily doses of
    2.5 mg induced withdrawal bleeding in 11 estrogen-primed amenorrhoeic
    women. 

    4.  EVALUATION

         The Committee concluded that the most appropriate end-point for
    evaluating the safety of residues of melengestrol acetate is the
    progestational effect in non-human primates. An ADI of 0-0.03 g/kg bw
    was established by applying a safety factor of 200 to the minimally
    effective dose of 5 g/kg bw per day of melengestrol acetate for
    affecting the menstrual cycle in female cynomolgus monkeys in a study
    over three menstrual cycles. This safety factor was used because the
    ADI is not based on a clear NOEL. As is its usual practice, the
    Committee rounded the value of the ADI to one significant figure.

    5.  REFERENCES

    Avenando, S., Tatum, H.J., Rudel, H.W. & Avenando, O. (1970) A
         clinical study with continuous low doses of megestrol acetate for
         fertility control.  Am. J. Obstet. Gynecol., 106, 122-127.

    Bollert, J.A. & Highstrete, J.D. (1969) U-21240 Depo-injectable:
         Teratology study in the rat. Upjohn Technical Report
         5301-69-7263013, 28 August 1969. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Bollert, J.A., Highstrete, J.D. & Andersen, S. (1970a) U-21240,
         depo-injectable; rat reproduction study. Upjohn Technical Report
         530170-7263-006, 29 April 1970. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Bollert, J.A., Highstrete, J.D., Andersen, S. & Hall, T. (1970b)
         U-21240 depo-injectable; teratology study in the rabbit. Upjohn
         Technical Report 5301-70-7263-017, 8 July 1970. Submitted to WHO
         by Upjohn Company, Kalamazoo, Michigan, USA.

    Carlson, R.G. (1968) U-21240; histology addendum to chronic dog
         toxicity report. Upjohn Technical Report 5301-68-7263-009, 27
         December 1968. Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Carlson, R.G. (1969) U-21240; histology addendum to chronic monkey
         toxicity report. Upjohn Technical Report 5301-69-7263-001, 22
         January 1969. Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Carlson, R.G. & Ceru, J.G. (1965) Oral LD50 in the rat. 21 December
         1965. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Carlson, R.G. & Hall, T.L. (1968) U-21240; chronic intramuscular
         toxicity in the dog; 12-month study with 6-month interim
         sacrifice. Upjohn Technical Report 5301-68-7263-007, 8 November
         1968. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Carlson, R.G. & Highstrete, J.D. (1968) U-21240; melengestrol acetate.
         Intraperitoneal LD50 in mouse and rat. Upjohn Technical Report
         22205-15-68-7330-009, 29 February 1968. Submitted to WHO by
         Upjohn Company, Kalamazoo, Michigan, USA.

    Casavilla, F., Stubrin, J., Maruffo, C., Van Nynanten, C. & Perez, V.
         (1970) Daily megestrol acetate for fertility control: A clinical
         study.  Contraception, 6, 361-372

    Charron, D.D., Goyings, L.S. & Kaczkofsky, H.W. (1973) Effect of MGA
         on mammary gland development in ICR and C3HAn/f mice. Upjohn
         Technical Report 623-9610-DDC-73-3, 15 June 1973. Submitted to
         WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Chenault, J.R., Kirton, K.T. & Roehm, P.A. (1990) U-21240: A subacute
         (one menstrual cycle), preliminary, oral dose-finding study for
         determination of the hormonal-no-effect level for melengestrol
         acetate (MGA) in the cynomolgus monkey. Upjohn Technical Report
         7224-90-024, 20 July 1990. Submitted to WHO by Upjohn Company,
         Kalamazoo, Michigan, USA.

    Chenault, J.R., Kirton, K.T., Roehm, P.A., Prough, M.J., Boucher, J.F.
         & Kokmeyer, J.W. (1993) U-21240 (melengestrol acetate
         [MGA(R)]): A chronic (180 day/6 menstrual cycle) oral study in
         sexually mature female cynomolgus monkeys to determine the
         hormonal-no-effect level of melengestrol acetate (MGA(R)).
         Upjohn Technical Report 7224-92-006, 30 September 1993. Submitted
         to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Clark, J.J. & Albert, H. (1962) U-21240 (6-dehydro-16-methylene
         Provera), subacute oral toxicity in the dog. 65 JJC, 29 June
         1962. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Clark, J.J., Albert, H. & Washburn, R.V. (1963) U-21240,
         17-hydroxy-6-methylenepregna-4,6-diene-3,20-dione,acetate.
         Teratogenic study in the rat. JJC No. 81, 22 January 1963.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Cooper, J.M. (1967) The metabkolism of megestrol acetate
         (17alpha-acetoxy-6-methylpregna-4,6-diene-3,20-dione) and its
         16-methylene derivative, melengestrol acetate
         (17alpha-acetoxy-6-methyl-16-methylpregna-4,6-diene-3,20-dione).
         PhD Thesis Dissertation, University of London, United Kingdom.

    Cooper, J.M., Elce, J.S. & Kellie, A.E. (1967) The metabolism of
         melengestrol acetate.  Biochem. J., 104, 57-58.

    Cornette, J.C. & Duncan, G.W. (1968) Effect of single intramuscular
         injections of melengestrol acetate (U-21240) and of
         medroxyprogesterone acetate (Provera, U-8839) on reproductive
         capabilities of rats. Upjohn Technical Report
         22205-15-687310-001, 29 February 1968. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Davis, R.A. (1973) Determination of MGA in heifer excreta. Unpublished
         report by Upjohn Company, Kalamazoo, Michigan, USA.

    Duncan, G.W., Lester, S.C., Hendrix, J.W., Clark, J.J. & Webster, H.D.
         (1964) Biological effects of melengestrol acetate.  Fertil. 
          Steril., 15, 419-432.

    Echternkamp, S.E. & Hansel, W. (1971) Plasma estrogens, luteinizing
         hormone, and corticoid in postpartum cows.  J. Dairy Sci., 54,
         800.

    European Agency for Evaluation of Medicinal Products (1996) Committee
         for Veterinary Medicinal Products: Medroxyprogesterone acetate.
         Summary report. EMEA/MRL/0129/96-Final. July 1996.

    Goyings, L.S. (1971a) Chronic oral safety and reproductive study in
         the cow with melengestrol acetate -- Part II, gross and
         histopathologic observations. Upjohn Technical Report
         610-9610-LSG-71-5, 30 March 1971. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Goyings, L.S. (1971b) The safety and reproductive performance on bulls
         of chronic orally administered melengestrol acetate -- Part II,
         gross and histopathologic observations. Upjohn Technical Report
         610-9610-LSG-71-6, 15 June 1971. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Goyings, L.S. (1973) MGA two-year tolerance and reproductive study in
         dogs, part two: Report on clinical, morphologic, and chemical
         pathologic findings. Upjohn Technical Report 610-9610-LSG-73-9, 5
         September 1973

    Goyings, L.S. & Kaczkofsky, H.W. (1969a) Effect of varying levels of
         MGA on the estrus cycle in the mouse (ICH-Upjohn strain). Upjohn
         Technical Report 610-9610-1, 17 March 1969. Submitted to WHO by
         Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S. & Kaczkofsky, H.W. (1969b) Thirty-day oral toxicity
         study of melengestrol acetate (MGA) in the mouse. Upjohn
         Technical Report 610-96102, 23 March 1969. Submitted to WHO by
         Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S. & Kaczkofsky, H.W. (1969c) Melengestrol acetate (MGA,
         U-21240) subacute toxicity study in the rabbit. Upjohn Technical
         Report 006-9610-25, 15 May 1969. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Goyings, L.S., Schwikert, R.S., Schul, G.A. & Blevins, D.I. (1966)
         Effect of melengestrol acetate (MGA) on the cow and its calf when
         administered during gestation and 35 days postpartum. Upjohn
         Technical Report 612-9610-1, 14 November 1966. Submitted to WHO
         by Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S., Kaczkofsky, H.W. & Carter, E. (1970a) MGA-500 liquid
         premix (corn oil) acute oral toxicity (LD50) in the rat. Upjohn
         Technical Report 615-9610LSG-70-4, 17 August 1970. Submitted to
         WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S., Kaczkofsky, H.W. & Carter, E. (1970b) U-21240 MGA-500
         liquid premix (propylene glycol) acute oral toxicity (LD50) in
         the rat. Upjohn Technical Report 615-9610-LSG-70-3, 17 August
         1970. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Goyings, L.S., Kaczkofsky, H.W. & Carter, E. (1970c) The acute dermal
         LD50 of MGA-500 liquid premix, corn oil (U-21240) in rabbits.
         Upjohn Technical Report 615-9610-LSG-70-6, 1 October 1970.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S., Kaczkofsky, H.W. & Carter, E. (1970d) The acute dermal
         LD50 of MGA-500 liquid premix, propylene glycol (U-21240) in
         rabbits. Upjohn Technical Report 615-9610-LSG-70-6, 1 October
         1970. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Goyings, L.S., Miller, C.C., Zimbelman, R.G. & Kaczkofsky, H.W. (1971)
         Carcinogenic study with melengestrol acetate (MGA(R)) in ICR
         mice. Upjohn Technical Report 610-9610-LSG-70-3, 4 October 1971.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Goyings, L.S., Geng, S., Kaczkofsky, H.W., Weddon, T.E. & Bogema, L.
         (1975) Melengestrol acetate (MGA(R)) oral teratology study in
         rabbits. Upjohn Technical Report 623-9610-LSG-73-2, 15 September
         1975, Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

    Goyings, L.S., Lauderdale, J.W. & Thomas, R.W. (1976) Melengestrol
         acetate (MGA) tolerance study with C3HAn/f female mice -- A
         second lifespan study. Upjohn Technical Report 623-9610-LSG-76-5,
         27 November 1976. Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Greenblatt, R.B. & Rose, F.D. (1962) Delay of menses: Test of
         progestational efficacy in induction of pseudopregnancy.
          Obstet. Gynecol., 19, 730.

    Harbach, P.R., Johnson, M.A. & Bhuyan, B.K. (1982) The V-79 mammalian
         cell mutation assay with melengestrol acetate (MGA(R), U-21240)
         with and without an S9 activation system. Upjohn Technical Report
         9610-82-7263-009, 8 December 1982. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Henricks, D.M., Dickey, J.F. & Hill, J.R. (1971) Plasma estrogen and
         progesterone levels in cows prior to and during estrus.
          Endocrinology, 89, 1350-1355.

    Hobson, W.C., Lauderdale, J.W., Zimbelman, R.G., Goyings, L.S., Knauf,
         V. & Kasson, C.W. (1976) Biological effects of MGA in the female
         monkey. 14 December 1976. Submitted to WHO by Upjohn Company,
         Kalamazoo, Michigan, USA.

    Kountz, S.L. & Wechter, W.J. (1977) Immunosuppression with
         melengestrol.  Transplant. Proc., 9, 1447-1453.

    Krzeminski, L.F., Byron, L., Cox, L. & Gosline, E. (1981) Fate of
         radioactive melengestrol acetate in the bovine.  J. Agric. Food 
          Chem., 29, 167-171.

    Lauderdale, J.W. (1970) Effect of approximately two years feeding of
         MGA on reproductive ability of bulls. Upjohn Technical Report
         610-9670-003, 18 December 1970. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Lauderdale, J.W. (1971b) Influence of melengestrol acetate on acute
         responses of the bovine uterus inoculated with  Escherichia 
          coli. Am. J. Vet. Res., 32, 1033-1038.

    Lauderdale, J.W. (1971a) Reproductive performance of cows and heifers
         fed melengestrol acetate during a two year interval -- Project
         610. Upjohn Technical Report 610-9670-004-JWL, 15 April 1971.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Lauderdale, J.W. (1973) Two year MGA tolerance and reproductive study
         in dogs. Upjohn Technical Report 610-9670-007, 8 March 1973.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Lauderdale, J.W. (1977a) Studies of a progestogen (MGA) as related to
         residues and human consumption.  J. Toxicol. Environ. Health, 
         3, 5-33.

    Lauderdale, J.W. (1977b) Effects of MGA(R) and grain on plasma
         concentrations of estrogens, corticoids, and progesterone for
         Holstein heifers. Upjohn Technical Report 623-9670-008, 9
         December 1977, Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Lauderdale, J.W. & Goyings, L.S. (1972) Melengestrol acetate (MGA)
         tolerance studies with ICR and C3HAn/f mice. Upjohn Technical
         Report 610-9610-LSG71-7, 7 June 1972. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Lauderdale, J.W., Goyings, L.S. & Kaczkofsky, H.W. (1972) Preliminary
         results for additional tolerance studies with C3HAn/f mice.
         Upjohn Technical Report 6239610-LSG-72-1 (623-9670-001), 20
         November 1972. Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Lauderdale, J.W., Goyings, L.S. & Kasson, C.W. (1980) Effect of dose
         of MGA(R) on mammary tumor development (lifespan) and serum
         prolactin concentration and mammary gland development (20 and 365
         days) in C3HAn/f female mice as influenced by a prolactin
         blocker--serum prolactin concentration for 20 day mice (substudy
         A). Upjohn Technical Report 623-9670-80-001, 6 March 1980.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Liggins, G.C. (1963) Treatment of endometrial carcinoma with a
         progestogen. Report of a case.  N.Z. Med. J., 62, 235-236.

    Liskin, L., Blackburn, R. & Ghani, R. (1987) Hormonal contraception.
         New longacting methods.  Popul. Rep., 15, 58-87.

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         Continuous progestagen contraception: A dose relationship study
         with chlormadinone.  Fertil. Steril.,18, 57-62.

    Mazurek, J.H. & Swenson, D.H. (1982) Evaluation of U-21240 in the
          Salmonella/microsome test (Ames assay) Upjohn Technical Report
         9610-827263-003, 30 August 1982. Submitted to WHO by Upjohn
         Company, Kalamazoo, Michigan, USA.

    Metzler, M. (1999) Metabolism and genotoxicity of the xenobiotic
         growth promoters zeranol, trenbolone acetate and melengestrol
         acetate: A critical review. Submitted.

    Neff, A.W. (1964) Oral melengestrol acetate (U21,240) tracer study No
         2 in the heifer. Unpublished report by Upjohn Company, Kalamazoo,
         Michigan, USA.. Not submitted.

    Nugent, C.A., Bressler, R., Kayan, S. & Worall, P. (1975) Suppression
         of cortisol by a progestational steroid, melengestrol.  Clin. 
          Pharmacol. Ther., 18, 338-344.

    Paterson, S.E. & Hall, C.C. (1983) 90-day oral toxicity study with
         MGA(R) and Monensin(R) in Sprague-Dawley rats. Upjohn
         Technical Report 528-9610-83001, 22 June 1983. Submitted to WHO
         by Upjohn Company, Kalamazoo, Michigan, USA.

    Petrow, V. (1967) Horizons in fertility control.  Med. J. Malaysia, 
         21, 294-306.

    Petzold, G. & Bedell, M. (1978) Evaluation of U-21240 in the DNA
         damage/alkaline elution assay. Upjohn Technical Report
         623-9610-78-002 (7263-78-7263021), 4 October 1978. Submitted to
         WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Phillips, T.J. (1966) Oral progestogens and adenocarcinoma of the
         uterus.  J. Obstet. Gynaecol. Br. Commonw., 73, 487-489.

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         endocrine influences on the growth and carcass quality of
         Holstein heifers.  J. Anim. Sci., 33, 836-842.

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         H.A .(1971b) Some carcass quality and endocrine criteria of
         Holstein heifers fed melengestrol acetate.  J. Anim. Sci., 32,
         628-635.

    Raczniak, T.J. (1982) Evaluation of melengestrol acetate (U-21240) in
         the primary rat hepatocyte unscheduled DNA synthesis assay (UDS)
         Upjohn Technical Report 623-9610-82-001, 28 July 1982. Submitted
         to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Raczniak, T.J., Wood, D.R., Kakuk, T.J., Skinner, P.J., Russell, K.B.,
         Lauderdale, J.W. & Thomas, R.W. (1981) One year effect of
         MGA(R) alone and in combination with
         6-methyl-8-ergoline-acetonitrile on serum prolactin values and
         mammary gland development in female C3HAn/f mice. Upjohn
         Technical Report 623-9610-81-001, 28 May 1981. Submitted to WHO
         by Upjohn Company, Kalamazoo, Michigan, USA.

    Raczniak, T.J., Wood, D.R., Hall, C.C. & Ash, K.A. (1983) Fertility
         and reproductive performance (segment I reproduction study) in
         Fischer/344 (F344/NUpj) rats after continuous dietary
         administration of melengestrol acetate (MGA) Upjohn Technical
         Report 623-9610-83-001, 26 January 1983. Submitted to WHO by
         Upjohn Company, Kalamazoo, Michigan, USA.

    Raczniak, T.J., Russell, K.B. & Ash, K.A. (1985) Chronic oral feeding
         study with melengestrol acetate (U-21240; MGA(R)) in female
         C3HAn/f mice -- A third lifespan study. Upjohn Technical Report
         7263-84-040, 18 January 1985. Submitted to WHO by Upjohn Company,
         Kalamazoo, Michigan, USA.

    Ray, J.A. & Ceru, J.G .(1969) U-21240; acute subcutaneous LD50 study
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         1969. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.

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          Carcinogenesis, 4, 239-241.

    Segaloff, A. (1965) Data transmitted by letter from Dr D.J. Taylor. 22
         September 1965. Submitted to WHO by Upjohn Company, Kalamazoo,
         Michigan, USA.

    Skinner, P.J., Goyings, L.S., Vanhuysen, C.N., Keener, S.K. & Russell,
         K.B. (1980) Third lifetime MGA mouse study: The effect of MGA
         given in conjunction with MEA on mammary gland development.
         Upjohn Technical Report 623-9610-80001, 31 January 1980.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Sokolowski, J.H. & Goyings, L.S. (1972) Two-year melengestrol acetate
         tolerance and reproductive study in beagles -- Interim report, 23
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         Michigan, USA.

    Sokolowski, J.H. & Van Ravenswaay, F. (1969a) Effects of melengestrol
         acetate on estrous inhibition in the female. U-21240 (MGA) in
         dogs. Upjohn Technical Report 618-9670-012, 18 March 1969.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Sokolowski, J.H. & Van Ravenswaay, F. (1969b) Effects of a low level
         of melengestrol acetate (MGA) on conception, pregnancy,
         parturition, and offspring in the female. Upjohn Technical Report
         9670-610-001, 28 July 1969. Submitted to WHO by Upjohn Company,
         Kalamazoo, Michigan, USA.

    Sywer, G.I.M. & Little, V. (1962) Action and uses of orally active
         progestational agents.  Proc. R. Soc. Med., 55, 861-868.

    Trzos, R.J. & Swenson, D.H. (1982) The micronucleus test with
         melengestrol acetate (U-21240). Upjohn Technical Report
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         Company, Kalamazoo, Michigan, USA.

    Walker, B.E. (1967) Induction of cleft palate in rabbits by several
         glucocorticoids.  Proc. Soc. Exp. Biol. Med., 125, 1281-1284.

    Webster, H.D. & Frielink, R. (1962a) Intraperitoneal LD50 in the
         mouse. 29 March 1962. Submitted to WHO by Upjohn Company,
         Kalamazoo, Michigan, USA.

    Webster, H.D. & Frielink, R. (1962b) Subacute oral toxicity in rats.
         4669-HDW: 147148, 6030-HDW: 29-30 and 37-39, 1 June 1962.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Wood, D.R., Raczniak, T.J., Hall, C.C. & Ash, K.A. (1983) A continued
         oral feeding study with melengestrol acetate in Fischer/344
         (F344/NUpj) rats exposed from conception to sexual maturity.
         Upjohn Technical Report 623-9610-83-002, 26 January 1983.
         Submitted to WHO by Upjohn Company, Kalamazoo, Michigan, USA.

    Zimbelman, R.G. & Smith, L.W. (1966a) Control of ovulation in cattle
         with melengestrol acetate. I. Effect of dosage and route of
         administration.  J. Reprod. Fertil., 11, 185-191.

    Zimbelman, R.G. & Smith, L.W. (1966b) Control of ovulation in cattle
         with melengestrol acetate. II. Effects on follicular size and
         activity.  J. Reprod. Fertil., 11, 193-201.

    Zimmer, D.M., Bhuyan, B.K. & Mazurek, J.H. (1978) Evaluation of
         U-21240 in the  Salmonella/microsome test (Ames assay). Upjohn
         Technical Report 623-789610-003 (7263-78-7263-023), 28 November
         1978. Submitted to WHO by Upjohn Company, Kalamazoo, Michigan,
         USA.
    


    See Also:
       Toxicological Abbreviations
       MELENGESTROL ACETATE (JECFA Evaluation)