ZERANOL
EXPLANATION
Zeranol was considered at the twenty-sixth meeting of the Joint
FAO/WHO Expert Committee on Food Additives (Annex 1, reference 59),
but it could not be evaluated at that time because the necessary
documentation on residue levels, good animal husbandry in relation to
the use of the agent, and details of methods of analysis were not
available. At the twenty-seventh meeting (Annex 1, reference 62), the
Committee provisionally accepted the use of zeranol as an anabolic
agent for the production of meat for human consumption in accordance
with good animal husbandry practice, and requested submission of the
results of (1) a study known to be in progress to establish a
no-effect level for hormonal activity in non-human primates and (2)
adequate carcinogenicity studies in two rodent species.
This monograph contains the data previously considered by the
Committee, as well as data that have been submitted recently.
BIOLOGICAL DATA
Toxicological studies
Special studies on carcinogenicity
Mice
Four groups of 50 male and 50 female CDI mice each were
maintained on diets containing 0, 0.15, 1.5, or 15 ppm zeranol for 104
weeks. Another group of 50 male and 50 female CDI mice, which served
as a positive control, were maintained on a test diet containing
2.5 ppm estradiol-17ß. Haematology studies on 10 males and 10
females/group were carried out at weeks 26, 52, and 78 and on all
surviving animals at week 104. At week 104 of the study all surviving
mice were killed and autopsied. Full histopathological examinations
were performed on all control, highest-dose zeranol, and estradiol-17ß
test animals, as well as on all animals dying during the course of the
study. Partial histopathological examinations (adrenals, kidneys,
liver, lungs, mammary gland, uterus, vagina, cervix, prostate, seminal
vesicles, testes, pituitary, skin, and all masses seen directly) were
performed on all other animals.
Mortality in the animals treated with zeranol was slightly higher
than in controls. However, mortality was markedly increased in the
animals fed estradiol-17ß, particularly in females. Male mice treated
with zeranol showed a slight decrease in body weight compared to
control mice during the first 52 weeks of the study, but the effect
was not apparent during the second 52-week period. In contrast, males
treated with estradiol-17ß showed a significant decrease in body
weight throughout the course of the study. Females on the test diets
showed similar weight gains to the controls. The levels of food
consumption of male and female mice on diets containing zeranol and of
males receiving estradiol-17ß were similar to controls. Females
receiving estradiol-17ß showed increased food consumption (from week
80) compared to the control animals.
During the course of the study, there were minor changes in
haematological parameters in the mice treated with zeranol. Dosing
with estradiol-17ß resulted in a reduction in haemoglobin levels and
red blood cell count in males, and changes in the differential white
blood cell patterns in females. At autopsy, a few significant changes
were observed in the mice fed 15 ppm zeranol, namely pituitary
enlargement and dilation of the seminal vesicles in males. In female
mice there was an increased incidence of alopecia. The incidences of
these changes were greatly increased in mice in the positive control
group. Histopathological examination of the highest-dose zeranol-
treated mice showed brown degeneration in the adrenals in both sexes
and conversion of the submaxillary salivary glands to the female type,
trabeculation in the sterum, and dilation of the seminal vesicles in
males. These estrogenic effects were very mild compared to those in
mice fed estradiol-17ß. In the 0.15 ppm, but not the 1.5 ppm, group
the incidences of ovaries with corpora lutea present and no secondary
follicles (females) and dilatation of the seminal vesicles (males)
were significantly decreased compared with controls. However, these
effects were not considered to be true hormonal effects, because (1)
the effect on the ovaries was not dose related and (2) the lower
incidence of dilatation of the seminal vesicles was more common in
older animals, and the lower incidence in treated animals probably was
related to the longer average survival time of the controls. Other
significant effects observed in the 15 ppm zeranol group included an
increase in mucous cervical and vaginal epithelium (20/46 compared to
10/48 in controls; 0/47 in the positive control group) and subcapsular
hyperplasia of the adrenals in males (30/49 compared to 16/50 in
controls). The only tumourogenic effect of zeranol noted in this study
was in the pituitary gland of males in the highest-dose group. The
incidences of anterior lobe adenomas and hyperplasia plus tumours in
the anterior lobe noted in this study are summarized in Table 1.
Table 1. Incidences of hyperplasia and rumours of the pituitary gland
in male mice
Negative Zeranol (ppm in the diet) Positive
Finding control 0.15 1.5 15 control
Anterior lobe 1 0 0 8 28
adenoma
Hyperplasia + 1 2 2 12 33
rumours in
anterior lobe
These tumours rarely occur spontaneously in mice. However,
neoplasia of the pituitary gland has been associated with the
administration of estrogen to certain strains of mice (Gardner, 1941),
and it is considered to be associated with hormonal imbalance
resulting from the administration of estrogenic hormones (Gardner,
1941, 1948).
In the estradiol-17ß group, the estrogenic effects were more
marked than those observed in mice treated with zeranol. These effects
included brown degeneration in the adrenals, increased trabeculation
in the sternum, conversion of male submaxilliary salivary glands to
the female type, shrinkage of seminal vesicles, scarcity of secondary
follicles and corpora lutea in the ovaries, uterine inflammation,
endometriosis and hyalinization, cervical adenosis, vaginal epithelial
keratinization, and mammary gland development. Tumourgenic effects in
the positive controls included anterior lobe tumours of the pituitary
gland and testicular interstitial cell tumours in males and mammary
gland carcinomas with metastasizing adenocarcinomas in females
(Everett, et al., 1987a).
Rats
Four groups of 50 male and 50 female Sprague-Dawley rats each
were maintained on diets containing 0 (negative control), 0.25, 2.5,
or 25 ppm zeranol for 104 weeks. Two other groups of 25 male and 25
female rats each, which served as positive dietary controls, were
maintained on diets containing either 2.5 ppm estradiol-17ß throughout
the study or 25 ppm estradiol-17ß for 8 weeks, which was reduced to
2.5 ppm for the rest of the experiment (toxicity was observed at
25 ppm). In another group of 25 females, which served as an implant
positive control group, each animal was implanted s.c. with
approximately 15 mg estradiol-17ß at the commencement of the study.
Haematology studies on 10 males and 10 females per group were
carried out at weeks 26, 52, and 78 of the study and on all surviving
rats at 104 weeks. Haematology, clinical chemistry analyses, and
urinalyses were carried out on 15 males and 15 females per group
(where available) at week 103 of the study. At week 104, all surviving
animals were killed and autopsied. Full histopathological examinations
were performed on all control, highest-dose zeranol, and estradiol-17ß
test animals. Partial histopathological examinations (adrenals,
cervix, ovaries, pituitary, prostate, seminal vesicles, testes, skin,
kidneys, liver, lungs, mammary gland, and masses) were carried out on
all other test animals. Animals dying during the course of the study
were autopsied and complete histological examinations were performed
on them.
Male rats in the highest-dose zeranol group showed a slight
decrease in weight gain compared to negative controls during the first
52 weeks of the study, but weight gains were similar to controls
during the remainder of the study. A slight decrease in body weight
was observed in males in the 0.25 ppm group and in females in the 0.25
and 2.5 ppm groups. Females in the highest-dose group showed similar
weight gains to controls, except during the first 90 days of the
study, when a reduction in body weight was observed. In contrast, rats
receiving estradiol-17ß showed a marked reduction in body weight, the
greatest depression being noted in females implanted with
estradiol-17ß. Levels of food consumption in the negative control and
groups fed zeranol were similar. There was some reduction in food
intake in rats in the positive control groups.
Haematological studies showed that female rats in the 0.25 ppm
zeranol group had increases in red blood cell parameters (haemaglobin
level and red blood cell count) and white blood cell counts compared
to controls. Animals in all other zeranol test groups had results
similar to negative controls. Female rats in the positive dietary
control groups showed elevated white blood cell counts, whereas those
implanted with estradiol-17ß showed decreases in red blood cell
parameters and increased neutrophil counts. There were no significant
inter-group differences in clinical chemistry or urinalysis results.
Survival of test animals was similar in the negative control and
zeranol test groups. In the positive dietary control groups, survival
was lower than in the zeranol groups, but most deaths occurred after
80 weeks of treatment. In the group implanted with estradiol-17ß,
deaths occurred after 20 weeks, and there were no survivors at week 44
of the study. No common causes of death were noted in the treated
animals receiving zeranol, although those receiving estradiol-17ß
showed a higher incidence of enlarged pituitaries than did controls.
At autopsy, organ weight analyses of rats fed zeranol showed no
significant differences between male rats and their respective
controls. There was an apparent increase in uterus weight in females
in the highest-dose group, but because of the large variation in ovary
weights in this group, this was not statistically significant. Male
rats receiving dietary estradiol-17ß had significantly-decreased
kidney weights, and females had reduced absolute ovary weights
compared to controls.
Histological examination of the tissues of the zeranol-treated
animals showed no compound-related increases in neoplasms.
Non-neoplastic findings in female rats included an increase in
dilatation of the uterus in the highest-dose group, as well as an
increase in stratified squamous epithelial cells of the cervix in the
2.5 and 25 ppm groups. There was an increase in mammary hyperplasia in
the 2.5 ppm group, but this was not statistically significant when an
age-adjustment analysis was carried out. Kidney tubular epithelial
haemosiderosis occurred more frequently in the 0.25 ppm test group
than in controls, but this was not considered to be a compound-related
effect.
All females treated with estradiol-17ß showed marked estrogenic
effects, with significant changes in the ovaries, uterus (including
squamous metaplasia and epithelial dysplasia), vagina and cervix, and
mammary glands. Effects were also reported in the spleen, sternum,
stomach, kidney, and lungs. In males treated with estradiol-17ß,
effects were observed in the liver, pancreas, mammary gland, heart,
testes, kidneys, and spleen. Histopathological changes in animals
treated with estradiol-17ß implants were more severe than those in
animals receiving estradiol-17ß in the diet. There was also a marked
increase in pituitary adenomas (type 3) in test animals in the implant
group compared with controls (Everett, et al., 1987b).
Special study on estrogenic potency
The uterotropic response in sexually immature rats was used to
compare the estrogenic potency of zeranol and its metabolites
zearalanone (the 7-keto oxidation derivative) and taleranol
(7-ß-hydroxy epimer of zeranol) with estradiol-17ß. Administration of
the test compound by the oral route showed the estrogenic potency of
zeranol, zearalanone, and taleranol to be 1/150, 1/400, and 1/350,
respectively, that of estradiol-17ß. By the s.c. route zeranol was
1/500 less estrogenic than estradiol-17ß (Everett et al., 1987c).
Special studies on no-hormonal effect levels
Monkeys
Eighteen female cynomolgus monkeys were bilaterally
ovariectomized. After an 11-week stabilization period, the animals
were assigned to 3 groups. The test groups were then dosed orally with
zeranol in 0.2% carboxymethylcellulose in a flavoured beverage at dose
levels equivalent to 0.05, 0.5, or 5 mg/kg b.w./day for 13 weeks. A
control group was not used. Routine clinical chemistry, haematology,
and urinalysis determinations were made one month prior to dosing, and
at 1 and 3 months during the test period. Serum levels of insulin and
thyroid stimulating hormone (TSH) were determined once prior to
dosing, and at 1 and 3 months during the dosing period. FSH was
determined on a weekly basis during the study through 4 weeks
post-dosing.
Clinical chemistry, haematology, and urinalysis parameters were
within the normal ranges. No compound-related effects were observed on
serum levels of insulin or TSH. FSH values during the 24 hours after
the first daily dose of zeranol showed some variation, but no biphasic
response was observed. FSH values measured during the course of the
study showed variations, but no consistent depression was observed.
Baseline measurements and measurements of maturation index of
vaginal epithelial cells during the course of the study indicated an
estrogenic effect in animals receiving 0.5 and 5 mg/kg b.w./day
zeranol. The effect was not apparent at 0.05 mg/kg b.w./day
(Singh et al., 1984a; CIC, 1985).
Three groups of 6 mature male cynomolgus monkeys each were dosed
daily with zeranol at levels equivalent to 0.05, 0.5, or 5 mg/kg
b.w./day for a period of 13 weeks. Routine clinical chemistry,
haematology, and urinalysis determinations were made in each of the
animals twice prior to dosing, then after 1 and 3 months of dosing.
Serum levels of insulin, FSH, and testosterone were also measured. At
the end of the dosing phase, testicular biopsies were taken and
examined microscopically.
Body-weight gain was normal during the test period. Clinical
chemistry, haematology, and urinalysis parameters were within normal
limits. Serum levels of insulin, TSH, and FSH showed no compound-
related changes. Testosterone levels were highly variable in
individual animals, but they showed no compound-related effects.
Examination of testicular biopsies showed no effects on the
spermatogonia, the primary and secondary spermatocytes, or the sperm.
The Leydig cells were normal (Singh et al., 1984b).
Five groups of 6 sexually mature adult intact female cynamologus
monkeys each were used for this study. Three groups were administered
zeranol orally on a daily basis for 3 menstrual cycles at dose levels
equivalent to 0.05, 0.5, or 5 mg/kg b.w./day. One group was dosed with
the vehicle (0.2% carboxymethycellulose in a fruit flavoured drink)
and the other with estradiol-17ß at a dose level equivalent to
0.01 mg/kg b.w./day. Haematology, clinical chemistry, and urinalysis
determinations were made prior to dosing, during the dosing phase, and
during a withdrawal period. Serum levels of insulin, TSH, FSH,
estradiol-17ß, and progesterone were measured during the course of the
study (every third of the menstrual cycle). Vaginal cornification
(maturation index) and daily observations were made for changes in sex
skin coloration or swelling.
No treatment-related effects on body weight were observed. Sex
skin changes during the cycles of dosing and withdrawal showed no
compound-related effects in animals treated with zeranol or
estradiol-17ß. None of the treatments had a significant effect on
clinical chemistry or haematology parameters or on serum levels of
insulin or TSH. There were no compound-related effects on serum levels
or cyclic behaviour of FSH, estradiol, or progesterone in any of the
test groups. There was no significant change in the type of vaginal
epithelial cells during the dosing phase of the study (Singh et al.,
1984c; Singh & Griffin, 1984).
Three groups of 8 mature female rhesus monkeys each were dosed
daily with zeranol at levels equivalent to 0.5, 5, or 50 mg/kg
b.w./day for three menstrual cycles, or 111 days, after a pretreatment
control cycle. A control group of 6 monkeys received the vehicle
(orange juice) used to suspend the zeranol. Daily blood samples were
taken from each animal during the last pretreatment control cycle,
every-other-day during the first two treatment cycles, and daily
during the treatment cycle. Serum concentrations of estradiol,
progesterone, LH and FSH were determined by radioimmunoassay.
The average menstrual cycle showed no significant compound-
related effects in the 0.5 or 5 mg/kg b.w./day group. By contrast, the
menstrual cycle in the high-dose group was completely inhibited during
the first 60 days of treatment, and markedly inhibited during the
third cycle. Ovulation rates were similar in the control, low-dose,
and mid-dose groups, but markedly inhibited in the high-dose group. No
differences were observed in the quantitative pattern of estradiol
secretion in the control and mid-dose groups; however, the pattern was
altered in the high-dose group, where estradiol levels were
significantly reduced. LH and FSH levels and patterns of secretion
were similar to those observed in the pretreatment cycle in the
control, low-dose, and mid-dose groups, but levels were high in the
high-dose group, and the pattern of secretion was different than in
the other groups (Hess, 1986).
Special studies on mutagenicity
The available mutagenicity data on zeranol and its metabolites,
zearalanone and taleranol, are summarized in Tables 2, 3, and 4.
Special studies on reproduction
Rats
Groups of 10 male and 10 female Sprague-Dawley rats, weighing
approximately 200 g each that were 7 weeks of age at the beginning of
the study, were maintained on diets containing 0, 0.25, 1.77, 12.5, or
25 ppm zeranol commencing 4 weeks prior to mating and continuing until
termination of the study. The offspring (F1 generation) were
maintained on the test diets for 3 to 4 weeks after weaning. F0
males and females were killed and necropsied after their respective
functions were completed. The F1-generation animals were killed and
necropsied at approximately 6 weeks of age.
The F0 males showed a reduction in body weight compared to
controls at all dose levels (19% in the high-dose group). Food
consumption of males fed zeranol was also reduced. Although F0
females fed zeranol showed only a slight reduction in food intake
compared to controls, those in the 12.5 and 25 ppm groups showed
significant reductions in body weight (21% in the high-dose group).
Indices of fertility and general reproduction were evaluated using the
fertility index, gestation index, live birth index, viability index
(days 0 - 4), and lactation index (days 4 - 21). There were no
significant dose-related effects. Male and female pups in the
high-dose group showed a significant reduction in body-weight gain
during the lactation period, and this was maintained during the
post-weaning period. Decreased weight gain was also observed in the
12.5 ppm group, but not in the lower-dose groups.
At autopsy, absolute and relative organ weight analyses of the
F0-generation animals showed decreased liver weight in all test
groups. All males fed zeranol showed reduced absolute and relative
seminal vesicle weights, and females had reduced absolute and relative
ovary weights. Gross necropsy of the animals from both generations did
not show any compound-related effects (Everett & Perry, 1984).
Table 2. Results of mutagenicity assays on zeranol
Test system Test object Concentration Results Reference
Ames test1 S. typhimurium 1 - 500 µg/plate Negative Bartholomew & Ryan,
TA98, TA100, 1980
TA1538
Ames test2 S. typhimurium 1 - 10000 µg/ Negative Jagannath, 1982a
TA100 plate
Forward Mouse lymphoma 25 - 600 µg/ml Negative Cifone, 1982
mutation L5178Y TK+/-
assay1 -3.7.2C cells
Bone marrow CD-1 mice 0.5, 1.5, 5 g/kg Negative Cimino, 1982
cytogenic
assay
Hepatocyte Adult male 1.3 × 10-3 to Negative Williams, 1983
primary F344 rats 1.3 × 10-5 mg/ml
culture DNA
repair assay
DNA binding Rat hepatocyte Not available Negative Williams, 1984
assay primary
cell culture
Rec-assay B. subtilis Not available Positive Scheutwinkel et al.,
H17, M45 1986
SOS-chromo E. coli Not available Negative Scheutwinkel et al.,
test2 PQ37 1986
V79/SCE Chinese hamster Not available Negative Scheutwinkel et al.,
test2 V79 cells 1986
1 With rat liver S-9 fraction.
2 Both with and without rat liver S-9 fraction.
Table 3. Results of mutagenicity assays on zearalanone1
Test system Test object Concentration Results Reference
Ames test2 S. typhimurium 50, 500, 1000 µg/ Negative Ingerowski et al.,
TA98, TA100, plate 1981
TA1535, TA1537
TA1538
Ames test3 S. typhimurium 1.0 - 10000 µg/ Negative Jagannath, 1982b
TA98, TA100, plate
TA1535, TA1537
TA1538
Forward Mouse lymphoma 3.13 - 300 µg/ml Negative Cifone, 1983
mutation L5178Y TK +/-
assay cells
Bone marrow CD-1 mice 0.5, 1.67, Negative Cimino, 1983
cytogenic 5 g/kg
assay
Hepatocyte Adult male 5 × 10-10 to Negative Williams, 1985a
primary F344 rats 5 × 10-4 mg/ml
culture DNA
repair assay
1 7-keto oxidation derivative of zeranol.
2 With rat liver S-9 fraction.
3 Both with and without rat liver S-9 fraction.
Special studies on teratogenicity
Mice
Pregnant CDI-1 mice were injected s.c. with log doses of zeranol
ranging between 10 and 10000 ug/kg b.w./day on days 6 - 10 of
gestation. Half of the mice were allowed to deliver, and the remaining
half were delivered by caesarian section. At doses of 1000 and above
the number of litters decreased from 81% in the controls to 35% in the
treated animals. A decrease in the average weight of the pups and an
increased number of stillborn litters were observed. Limited
macroscopic examination showed increased medullary trabeculae in the
bones (Davies et al., 1977).
Rats
Groups of 4 female pregnant rats received zeranol orally in
single doses of 4 mg on days 1, 2, 3, 4, and 5 of gestation, 1 mg/day
during days 1 - 4 of gestation, or a single dose of 8 mg on day 6 of
gestation. The rats were killed on day 9 of gestation. Zeranol
appeared to inhibit implantation up to day 4 of administration. In
experiments in which implantation occurred, the fetuses had begun to
resorb by day 9 (IMC, 1980a).
Table 4. Results of mutagenicity assays on taleranol1
Test system Test object Concentration Results Reference
Ames test2 S. typhimurium 1 - 500 µg/plate Negative Bartholomew & Ryan,
TA98, TA100 1980
TA 1548
Ames test3 S. typhimurium 1 - 5000 µg/plate Negative Jagannath, 1983
TA100
Forward Mouse lymphoma 20 - 160 µg/ml Borderline Cifone, 1985
mutation L5178Y TK+/-
assay2 -3.7.2C cells
Bone marrow ICR mice 8.5 - 85 mg/ml Negative Ivett, 1985a
cytogenic
assay
Hepatocyte Adult male 2 × 10-5 to Negative Williams, 1985b
primary F344 rats 2 × 10-1 mg/ml
culture DNA
repair assay
Chromosomal Chinese 12.5 - 250 µg/ml Positive Ivett, 1985b
aberration hamster ovary (- act.)
frequency cells Negative
assay3 (+ act.)
Dominant H/a (ICR)BR 0.5, 1.5, 5.0 Negative Brusick & Myhr,
lethal assay mice g/kg b.w./day 1986
1 7ß-hydroxy epimer of zeranol.
2 With rat liver S-9 fraction.
3 Both with and without rat liver S-9 fraction.
A three-generation study in rats was performed in which males
weighing 69 - 113 g each and females weighing 65 - 109 g each were fed
zeranol in the diet at levels of 0, 0.01, 0.10, or 0.20 ppm for each
successive generation, each comprising three consecutive litters.
Animals in each generation received zeranol in the diet for 70 days
prior to mating, they were removed from the diet from day of delivery
of the dams throughout lactation until weaning, and then they were
reinstated at the same levels as at the start of the study. Pups from
the Fb litters were used for the reproduction of the subsequent
generation; the pups from the Fc litters were used for the
teratogenicity study. While there were some differences in the litter
data for the three consecutive generations which appeared to be
significant, there was no trend or dose-response relationship. No
adverse effects were observed which could be attributed to treatment
with zeranol (Wazeter & Goldenthal, 1976).
Zeranol, administered orally at doses of 0.312, 1.25, or
5.0 mg/kg b.w./day to male rats for 60 days prior to mating with
untreated females, induced delayed insemination upon cohabitation and
delayed initiation of conception in the females (Williams, 1982).
Zeranol, administered orally at doses of 0.312, 1.25, or
5.0 mg/kg b.w./day to female rats for 60 days prior to mating with
untreated males, caused a decrease in the litter size, an increase in
stillbirths, and a decrease in neonate survival (Williams, 1982).
Pregnant female rats were administered orally 2 or 6 mg/kg
b.w./day zeranol from days 6 - 15 of gestation. At laparotomy on day
20 of gestation, reduced numbers of live fetuses and increased numbers
of resorptions were noted (Williams, 1982).
Rabbits
Zeranol was administered orally at doses of 1 or 5 mg/kg b.w./day
to pregnant does on days 6 - 18 of gestation. Fetuses were removed by
laparotomy on day 28. No abnormalities were noted (Williams, 1982).
Acute toxicity
Species Sex Route LD50 Reference
(g/kg b.w.)
Mouse ? oral > 40 IMC, 1980b
F i.p. 4.4 IMC, 1980b
Rat ? oral > 40 IMC, 1980b
F i.p. 9-11 IMC, 1980b
Groups of 25 rats/sex/dose received by oral gavage 0 or
200 mg/kg b.w. zeranol, and they were sacrificed 24 hours after
dosing. There was a decrease in blood glucose levels in all treated
rats, and an increase in total cholesterol in the males and a decrease
in the females. There was a decrease in the weight of the epididymis
of the males, and an increase in uterine weight in the females (Albany
Medical Collage, 1980).
Short-term studies
Mice
Groups of 20 CD-1 mice equally divided by sex were maintained on
diets containing 0, 1, 5, 25, 50, or 100 ppm zeranol for 8 weeks. No
deaths or clinical effects were observed during the course of the
study. There were no significant differences in body weights, food
consumption, or water consumption between test and control groups. At
autopsy, no compound-related gross lesions were observed. No
compound-related differences were observed in absolute or relative
organ weights between test and control groups of the liver in males
and females or of the testes in males. However, changes were observed
in the absolute and relative weights of the ovaries (decreased) at
dose levels of 25, 50, and 100 ppm zeranol and of the uterus
(increased) at 100 ppm zeranol in females (Perry & Everett, 1984).
Rats
Groups of 40 Sprague-Dawley rats equally divided by sex were
maintained on diets containing the equivalent of 0, 0.02, 0.18, 1.2,
or 8.8 mg/kg b.w./day zeranol for a period of 13 weeks. Body weights
and food consumption were determined weekly. Haematological, clinical
chemistry, and urinalysis studies were carried out at weeks 5 and 13.
Ophthalmoscopy was carried out at week 10. At termination of the
study, gross observations and organ weight determinations were made on
all animals, and histopathological examinations were performed on the
principal organs and tissues from 10 animals of each sex in the
high-dose and control groups.
Six deaths occurred during the course of the study, which did not
appear to be compound-related. Only minor decreases in food
consumption were observed in the test groups compared to the control.
However, there was marked reduction in weight gain compared to
controls in the high-dose group, particularly in males. No compound-
related changes were observed in haematological, clinical chemistry,
or urinalysis parameters. Organ-weight analysis showed a slight
increase in relative liver weights in both males and females and in
the kidneys of rats in the high-dose group. Histological examination
showed an increased incidence of vacuolation in hepatocytes from both
males and females in the high-doss group as well as fat accumulation
in these cells. The kidneys of males in the high-dose group showed a
significant increase in calcified casts. These effects were only
marginal in the 1.2 mg/kg b.w./day group. No compound-related effects
were observed at 0.18 mg zeranol/kg b.w./day (Everett et al., 1983).
Twenty intact juvenile female rats were implanted s.c. with 12 mg
pellets of zeranol and sacrificed after 150 days. Five control animals
were used. Decreased growth, cysts in the ovaries, mammary glands, and
other parts of the body, decreased ovarian and uterine weights,
absence of corpora lutea, and marked squamous metaplasia of the
uterine mucosa were noted (Huis in't Veld & Kroes, 1974).
Groups of 30 - 35 rats/sex/group received by oral gavage 0 or
200 mg/kg b.w./day zeranol for 4 days. There were decreases in total
cholesterol and blood glucose levels, decreases in weights of the
testes, seminal vesicles, and epididmes of males, increases in weights
of the adrenals of males, and increases in weights of the uterus and
adrenals of females. Microscopically, the ovaries showed prominent
corpora lutea, and the testicular tubules contained spermatogonia but
no adult or mature spermatozoa. The lymphoid tissue contained a number
of large multinucleated cells (Albany Medical College, 1980).
Groups of 5 albino Manor Farm rats/sex/dose received by oral
gavage 25, 50, 100, 200, or 400 mg/kg b.w./day zeranol 5 days/week for
6 weeks. A control group was not included. During the 4th week of the
study, the 100 mg dose was increased to 800 mg/kg b.w./day and the
200 mg dose was increased to 1600 mg/kg b.w./day. Effects were
observed in all treatment groups that included changes in external
genitalia (reduction in the size of the testes in males and
enlargement of the vulva in females). Irritability, flaccidity,
alopecia, excessive micturition, and reduced growth rate were also
noted. After microscopic examination, changes noted were spermatogenic
arrest, atrophy of the prostate, seminal vesicles, and coagulating
glands, absence of graafian follicles, endometrial hyperplasia,
squamous metaplasia of the seminal vesicles, and calcified casts in
the kidneys (IMC, 1980c).
Three groups (number not given) of male and female Sprague-Dawley
rats received s.c. 0, 2.25, or 9.0 mg/kg b.w./day zeranol for 14 days.
The growth rate was reduced in the treated animals. There were
decreases in the relative weights of the gonads, prostate, and thymus
in males and of the gonads and thymus in females. Additionally, there
were increases in the relative weights of the seminal vesicles and
adrenals in males and of the uterus and adrenals in females. A
significant reduction in the number of corpora lutea was also noted
(IMC, 1980d).
Groups of male and female rats received 0.25, 1.25, or 6.25 mg/kg
b.w./day zeranol in the diet for 13 weeks. Slight growth suppression
at the two highest doses was observed (Williams, 1982).
Groups of male and female rats were administered 0.1, 0.8, or
6.4 mg/kg b.w./day zeranol for 26 weeks. Reduced food intake and
body-weight gains, a tendency toward lower haemoglobin values in
males, and mild hepatic changes, including irregularity in hepatic
cell size, were noted in females at the highest dose (Williams, 1982).
Dogs
One dog/sex/dose received orally by capsule 25, 50, 100, 200, or
400 mg/kg b.w./day zeranol 5 days/week for 6 weeks. During the 4th
week of treatment the 100 mg dose was increased to 800 mg/kg b.w./day
and the 200 mg dose was increased to 1600 mg/kg b.w./day. No control
animals were included. Beginning with the second week of treatment,
all females showed swollen vulvae. All males showed a decrease in
testicular size. At a dose of 100 mg/kg b.w./day and above, males and
females showed slight to marked increases in leucocytes and relative
decreases in lymphocytes. At 200/1600 mg/kg b.w/ day, there were
slight decreases in haemaglobin and haematocrit values, and increases
in erythrocyte sedimentation rates. Microscopically, mammary ductal
proliferation was noted in both males and females, vulval swelling
with cornification of vaginal epithelium and ovarian atrophy was
observed in females, and atrophy of the seminiferous epithelium and
prostate and hyperplasia or squamous metaplasia of the prostatic
urethra were noted in males. At doses of 400 mg/kg b.w./day and above,
there were increases in bone marrow cellularity and in the
myeloid/erythrocyte ratio (Williams, 1982).
Groups of male and female dogs received orally by capsule 0.25,
1.25, or 6.25 mg/kg b.w./day zeranol for 14 weeks. Following the
thirty-first dose, the 0.25 mg dose was increased to 12.5 mg/kg
b.w./day. There was a trend toward a reduction in the size of the male
sex organs and an increase in uterine weight. Microscopically, there
was an arrest in spermatogenesis, and prostatic epithelial atrophy was
seen in one male at 12.5 mg (Williams, 1982).
Groups of male and female dogs were fed diets containing 10, 100,
or 1000 ppm zeranol for 29 weeks. At 1000 ppm, noted changes included
weight loss in one male, rapid sedimentation rates, reduced
haemoglobin, increased leucocytes, and reduced lymphocytes in 3 males.
Microscopically, changes noted in the 1000 ppm group included
testicular atrophy, squamous metaplasia of the prostate, endometrial
hyperplasia, and ovarian atrophy. Hypercullularity of the bone marrow
and slight squamous metaplasia of the bladder were also noted
(Williams, 1982).
Long-term studies
Rats
Groups of 25 - 35 male and female rats were fed 0, 0.1, 0.8, or
6.4 mg/kg b.w./day zeranol for one year. During the 36th week of the
study the 0.1 mg/kg b.w./day dose level was increased to 20 mg/kg
b.w./day. Body-weight gain was depressed in all the treated animals.
The rats in the 0.1/20 mg/kg b.w./day group showed depressed
haemoglobin, increased thrombocyte counts, increased prothrombin time,
decreased absolute and relative weights of the prostate, ovary, and
seminal vesicles, and increased absolute and relative weights of the
uterus and pituitary. Hepatic cell glycogen depletion and irregularity
in hepatic cell size were observed in male rats only. Nephron
degenerative changes, slight to moderate atrophy of the ovary, seminal
vesicles, and prostate, slight reduction in the number of mature
spermatozoa, and instances of hypocellularity of the bone marrow were
also noted in this group of animals. Marked degenerative changes were
seen in the livers of the animals which died during the study
(Williams, 1982).
Groups of 25 Charles River CD rats/sex/dose were fed 0, 0.1, 0.8,
or 6.4 mg/kg b.w./day zeranol for 104 - 105 weeks. During the 28th
week of the study the 0.1 mg/kg b.w./day dose level was increased to
20 mg/kg b.w./day. Body-weight gain was depressed in all the treated
animals in the 6.4 and 0.1/20 mg/kg b.w./day dose groups. Greater
frequency of alopecia was observed in the two highest dose groups, and
one rat in the 6.4 and 4 rats in the 0.1/20 mg/kg b.w./day dose groups
developed cataracts during the last quarter of the study. Haemoglobin
and haematocrit values were reduced at 0.1/20 mg/kg b.w./ day, which
was more severe in females than in males. The animals in the
0.1/20 mg/kg b.w./day group had lower absolute weights of the seminal
vesicles, testes, ovaries, and prostate. Macroscopically, the findings
were those commonly noted in aging animals. Microscopically, changes
were seen in the liver, ovaries, uterus, testes, prostate, and seminal
vesicles of all the rats treated with zeranol. The changes in the
liver included hepatic cell depletion, irregularity in hepatic cell
size, parenchymal cell vacuolation, chronic inflammatory cell
infiltrates, and occasional instances of nodular hyperplasia; in the
ovaries, prostate, seminal vesicles, and testes, slight to moderate
atrophy was noted; in the uterus the observed changes included
endometritis, squamous metaplasia, and cystic hyperplasia of the
endometrium (Woodard, 1968).
Dogs
Groups of male and female dogs were fed 0.025, 2.5, or 25 mg/kg
b.w./day zeranol for one year. Slight testicular atrophy, moderate
prostatic atrophy, and squamous metaplasia were observed at the middle
dose. At the high dose, observed changes included increased
thrombocytes, decreased lymphocytes, rapid sedimentation rates,
elevated leucocyte counts, and marked cornification of the vaginal
epithelial cells. At terminal sacrifice, treatment-related changes in
the 25 mg/kg b.w./day group included marked atrophy of the gonads,
squamous metaplasia and inflammatory changes in the prostate and
uterus, squamous metaplasia of the urinary bladder epithelium, an
apparent increase in the myeloid elements of the bone marrow, and
endometrial hyperplasia (Williams, 1982).
Groups of 4 beagle dogs/sex/dose were fed 0, 1, 100, or 1000 ppm
zeranol in the diet for 104 weeks. Body weights of the male dogs in
all treatment groups were slightly higher than those of the controls.
Most of the observed changes were noted at the highest dose.
Haematological changes included rapid sedimentation rates, high
leucocyte counts, and reduced lymphocytes. Increased relative weights
of the prostate and pituitary and decreased relative weights of the
gonads in males, increased uterine relative weights in females, and
increased relative weights of the liver, kidneys, adrenals, and
thyroid of both males and females at the highest dose were noted.
Macroscopically, observed changes included enlarged mammary glands,
small testes, edematous prepuces, and enlarged vulvae, prostate, and
uteri. Microscopically, marked atrophy of the gonads, especially the
ovaries (which were reduced to cysts), squamous metaplasia and
inflammatory changes in the uterus and prostate, and chronic
inflammatory changes in the bladder were observed. Two males and one
female in the 100 ppm group had varying degrees of myeloid hyperplasia
and one male had marked atrophy of the bone marrow (Woodard, 1968).
Groups of 16 female beagle dogs were administered orally by
capsule 0, 15, or 38 mg/kg b.w./day zeranol in a cyclic manner
(21 consecutive days of dosing followed by 7 days off the drug) for a
period of seven years (91 cycles). Two high-dose and two low-dose dogs
died during the third year, and 3 low-dose dogs died or were
sacrificed in a moribund state during the fourth year of the study.
Clinical signs of toxaemia and gross lesions associated with
pyometritis were noted in these dogs. To prevent further deaths
related to pyometritis all surviving dogs were hysterectomized at
3 - 3.5 years into the study. Prior to hysterectomy, an increased
incidence of anorexia and lethargy were noted in the treated animals.
Mean body weights were slightly lower than controls in the 15 mg/kg
b.w./day group; they were significantly lower than controls in the
high-dose group. Haematological changes included a slight but
consistent reduction in absolute and relative adrenal weights in the
animals in the low-dose group at the four year interim sacrifice. At
terminal sacrifice, significant increases in mean and relative ovarian
weights of the animals in both treated groups were noted. Zeranol had
a relatively strong uterotropic effect and caused a sharp increase in
the proliferation and cornification of the vaginal, cervical, and
vulvar mucosa, cystic endometrial hyperplasia, endometriosis interna,
and pyometritis (Hogan, 1981a).
Monkeys
Groups of 16 sexually mature female rhesus monkeys (Macaca
mulatta) were administered orally by gavage 0, 15, or 75 mg/kg
b.w./day zeranol in a cyclic manner (21 days of consecutive dosing and
7 days off the drug) for a period of 10 years (131 cycles). Control
animals were administered the vehicle, which was a mixture of ethanol
and methylcellulose. Interim sacrifice was performed at one year
(2 animals) and at 2 and 4 years (4 animals each).
Seven animals died during the first nine months of causes
unrelated to treatment and were replaced to maintain the number of
animals per group. Ophthalmological changes included bilateral
hypopigmented foci in the macular and perimacular regions of the
retina, grainy appearance of the macula, and diminution or loss of the
macular reflex. Palpable mammary nodules and/or enlarged axillary
lymph nodes were seen sporadically in some of the animals. Palpable
uterine masses were observed in the high-dose animals at a markedly
higher incidence than in the control and low-dose groups. Prolonged
periods of amenorrhea were observed in the high-dose animals, which
was followed, in later years of the study, by prolonged periods of
vaginal bleeding. The treated monkeys gained less weight than the
controls throughout the study; at termination the mean body weight of
females at 15 mg/kg b.w./day was 15% lower, and that of females at
75 mg/kg b.w./day was 25% lower, than body weights of controls.
During the first 1.5 years of the study there were transient
reductions in mean haemaglobin concentrations and haematocrit and
erythrocyte values in the treated animals as compared to the controls.
Sporadic increases in the measured clotting time were noted throughout
the study. No other haematological changes were noted. Sporadic
increases in SGPT activity were noted at 15 mg/kg b.w./day, while
consistently higher SGPT activities were observed at 75 mg/kg
b.w./day. A similar pattern was observed with serum triglyceride and
cholesterol concentrations.
The animals in the high-dose group had consistently higher mean
absolute and relative weights of the liver and uterus and lower mean
absolute and relative ovarian weights than the controls. Similar
observations, but to a lesser degree, were noted in the animals fed
15 mg/kg b.w./day zeranol. At terminal sacrifice, the mean relative
weight of the pituitary and mean absolute and relative adrenal weights
of the animals in the high-dose group were higher than those of the
controls. No treatment-related macroscopic changes were noted at the
1- and 2-year interim sacrifices. At A years, there were dose-related
increases in the incidence and degree of endometrial proliferation. At
10 years, treatment-related changes included enlarged uteri, thickened
endometrium and myometrium, enlarged oviducts, and endometriosis
externa.
No treatment-related microscopic changes were noted at one year
into the study. At two years, one benign adenoma was noted in one
animal and a relative increase in mammary acinar tissue and duct
development were noted at 75 mg/kg b.w./day zeranol. At 4 years,
treatment-related changes in the high-dose group included endometrial
hyperplasia with occasional cystically dilated glands (all 4 animals),
absence of mature follicles and corpora lutea, superficial atrophy of
the cervical epithelium with squamous metaplasia of the basilar
portions of cervical glands, and increased lobular development and
mild ductular epithelial proliferation. At 10 years, treatment-related
changes were noted in most of the animals in the high-dose group that
included hyalization of the subepithelial connective tissue in the
vagina, squamous metaplasia of the basilar portions of the cervical
glands, ovarian atrophy and absence of corpora lutea, and marked
ductal and acinar hyperplasia of the mammary gland. Cystic endometrial
hyperplasia, myometrial hypertrophy, and endometriosis externa were
noted in all the animals at 75 mg/kg b.w./day zeranol and in three
animals in the low-dose group. Leiomyomas of the uterus/cervix
(fibroids) were present in 1/12, 2/7, and 2/14 animals in the control,
low-, and high-dose groups, respectively (Hogan, 1981b).
COMMENTS
The toxicological data available to the Committee included
results from the requested studies, as well as mutagenicity,
reproduction, and teratogenicity data.
Zeranol was shown to be a weak estrogen in long-term studies in
mice, rats, dogs, and monkeys. Most of the changes noted occurred in
mammary glands and organs of the reproductive tract. Zeranol did not
cause changes in other reproductive parameters in rats, and was not
teratogenic in mice or rats. Zeranol and its metabolites zearalanone
and taleranol were not mutagenic in a number of tests in bacterial and
mammalian systems. Zeranol (at an unspecified concentration) gave a
positive result in the Rec-assay (Bacillus subtilis) and taleranol
gave a positive result in the test with Chinese hamster ovary cells in
the absence of activation, but a negative result with activation.
In the carcinogenicity study performed in rats, dietary levels of
zeranol up to 25 ppm (equivalent to 1.25 mg/kg b.w./day) had
estrogenic but not carcinogenic effects. In the study performed in
mice, zeranol had significant estrogenic effects in male mice in the
highest dose group, receiving zeranol at 15 ppm in the diet
(equivalent to 2.25 mg/kg b.w./day); these mice also showed a higher
incidence of anterior lobe tumours of the pituitary gland than did
mice in the negative control group. Such tumours rarely occur
spontaneously in mice but are known to result from administration of
estrogenic hormones. The positive control group receiving a diet
containing 2.5 ppm estradiol-17ß showed a higher incidence of anterior
lobe tumours of the pituitary than did either animals receiving
zeranol or animals in the negative control group. Thus the Committee
concluded that the carcinogenic effect of zeranol was associated with
its estrogenic properties, and that the determination of a
no-hormonal-effect level for tumours would permit an estimate of safe
levels of exposure to be made.
A no-hormonal-effect level could not be established in male
cynomolgus monkeys since no estrogenic effects were observed even at
the highest dose administered (5 mg/kg b.w./day). In intact female
cynomolgus and rhesus macaque monkeys, 5 mg/kg b.w./day was
established as a no-hormonal-effect level. In ovariectomized female
cynomolgus monkeys the no-hormonal-effect level was 0.05 mg/kg
b.w./day. The Committee concluded that this model could be relevant to
the human population and, since the ovariectomized female cynomolgus
monkey is highly sensitive to estrogenic substances, adopted a
conservative approach by using this study as a basis for setting an
ADI for human beings.
EVALUATION
Level causing no hormonal effect
Monkey: 0.05 mg/kg b.w./day.
Estimate of acceptable daily intake
0 - 0.5 µg/kg b.w.
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See Also:
Toxicological Abbreviations
ZERANOL (JECFA Evaluation)