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 µg/kg expressed as 3H-melengestrol
acetate equivalents) and in the contents of the jejunum (4.4 µg/kg)
and large intestine (7.9 µg/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 µg/kg as 3H-melengestrol acetate
equivalents) and fat (7.7 µg/kg). In excretory organs, the highest
concentrations were found in the wall of the digestive tract (2-11
µg/kg), followed by salivary glands (3 µg/kg) and kidney (1.6 µg/kg).
3H-Melengestrol acetate equivalents were detected at concentrations
> 2 µg/kg in mammary glands, oviducts, adrenal glands, and thymus but
at about 1 µg/kg (e.g. 0.7 µg/kg in muscle) in all other tissues. The
limit of detection was 0.5 µg/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 µCi). 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 µg/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-8ß-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 µg/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 µg/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 µg/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
µg/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
µg/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 µg/kg bw per day, one at 5 µg/kg bw
per day, and two at 10 µg/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
µg/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 µg/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 µg/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
µg/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 µg/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 µg/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-17ß 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 µg/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 µg/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 µg/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 µg/mouse of
the prolactin inhibitor MEA; however, the dose given (1.9-49 µg/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 µg/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 µg/kg bw (three
males and 20 females), or 2 µg/kg bw (three males and 10 females) for
2 years and at 8 µg/kg bw per day (three males and 10 females) for 1
year followed by 4 µg/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 µg/kg bw per day and at
the subsequent dose of 4 µg/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
µg/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 µg/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 µg/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 µg/kg of diet, and none or one at
higher doses. The fertility and pregnancy rate of dams were slightly
decreased at 500 µg/kg of diet but were not significantly different
from controls, but only one animal at 1000 µg/kg of diet became
pregnant. In 10 selected inseminated animals from the control group
and those at 500 µg/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 µg/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 µg/kg of diet and significantly decreased the progesterone
concentration at doses > 2000 µg/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 µg/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 µg/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 µg/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 µg/animal per day,
equal to 1, 5, 10, 20, 40, and 80 µg/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 µg/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 µg/animal to 157 days at 400 µg/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 µg/plate ± Negative No Metabolic activation Zimmer et al. (1978)
TA98, TA100, system not specified
TA1537
Reverse mutation S. typhimurium 250-2000 µg/plate ± Negative Yes Mazurek (1982)
TA98, TA100,
TA1535, TA1537,
TA1538
Forward mutation V79 cells (hprt 2.5-10 µg/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 µg/ml Negative Yes Cytotoxic at doses Raczniak (1982)
(unscheduled hepatocytes > 500 µg/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 µg/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 µg/kg bw
(three males and 20 females), or 2 µg/kg bw (three males and 10
females) for 2 years and at 8 µg/kg bw per day (three males and 10
females) for 1 year followed by 4 µg/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 µg/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 µg/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 µg/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 µg/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 µg/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 µg/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 µg/kg bw per day did not
suppress plasma cortisol concentrations, while 5 µg/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 µg/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 µg/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 µg, and the
maximal effect appeared to occur at 300-400 µg, 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 µg/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 µg/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 µg/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-17ß 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 µg/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 µg/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 µg/kg bw per day and one at 10 µg/kg
bw per day failed to ovulate during treatment. Monkeys at 2.5 and 10
µg/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
µg/kg bw per day, and luteinizing hormone was suppressed at 2,5 and 5
µg/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 µg/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 µg/kg bw per day and one at 10
µg/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 µg/kg bw per day and of progesterone at 25
µg/kg bw per day. Significantly fewer animals at the highest dose
menstruated and ovulated, and significantly more animals at 10 and 25
µg/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 µg/kg bw per day,
although not statistically significant, were consistent with the
hormonal response seen at higher doses. The Committee considered that
5 µg/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 µg/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 µg/kg bw
per day consistently suppressed estrus in all animals. Melengestrol
acetate was also fed to heifers at a dose equal to 1.8 µg/kg bw per
day from 2.5 through 11.3 months of age. When the animals reached
maturity, the serum concentrations of estradiol-17ß 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 µg 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 µg/kg bw per day for 2 years or at
8 µg/kg bw per day for 1 year followed by 4 µg/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 µg/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
µg/kg bw per day, or (to males only) at 8 µg/kg bw per day for 2
years, or (to females only) at 8 µg/kg bw per day for 1 year followed
by 4 µg/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 µg/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 µg/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 µg/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 µg/kg bw
was established by applying a safety factor of 200 to the minimally
effective dose of 5 µg/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.
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See Also:
Toxicological Abbreviations
MELENGESTROL ACETATE (JECFA Evaluation)