50, 271–279 (1999)
Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Maternal and Developmental Toxicity Evaluation of Melatonin
Administered Orally to Pregnant Sprague-Dawley Rats
G. Jahnke,* ,1 M. Marr,‡ C. Myers,‡ R. Wilson,† G. Travlos,† and C. Price‡
*Reproductive Toxicology Group and †Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709; and ‡Center for Life Sciences and Toxicology, Research Triangle Institute,
Research Triangle Park, North Carolina 27709
Received on November 12, 1998; accepted on April 7, 1999
Melatonin (MEL) is a widely used, over-the-counter sleep aid,
and it has putative contraceptive, antioxidant, antiaging, and
anticancer effects. The developmental toxicity potential for repeated oral doses of MEL had not previously been evaluated. In
the present studies, time-mated, Sprague-Dawley-derived (CDt)
rats were administered MEL or vehicle by gavage on gestation
days (gd) 6 –19. MEL-treated groups received 1-, 10-, 100-, 150-, or
200-mg/kg body weight/day in the screening study (15 rats/group),
and 50, 100, or 200 mg/kg/day in the definitive study (25 rats/
group). In both studies, maternal food/water consumption, body
weight, and clinical signs were monitored at regular intervals
throughout gestation. At termination (gd 20, both studies), maternal liver and gravid uterine weights, number of ovarian corpora
lutea, conceptus survival, fetal sex, and fetal body weight were
evaluated. Fetal morphological examination included external
structures (both studies) as well as visceral and skeletal structures
(definitive study). In the screening study, maternal serum levels of
17b-estradiol, progesterone, prolactin, and luteinizing hormone
were determined by radioimmunoassay, and mammary tissue was
fixed, stained, and evaluated for percent glandular area within the
fat pad. No maternal morbidity/mortality was found in either
study. In the screening study, aversion to treatment (>100 mg/kg/
day) and reduced maternal weight gain (>150 mg/kg/day) were
noted, but reproductive/endocrine parameters and fetal development were not affected. In the definitive study, aversion to treatment was noted at >50 mg/kg/day, and mild sedation, reduced
maternal food intake, and reduced body weight gain were found
during initial treatment with 200 mg/kg/day. MEL had no effect
on prenatal survival, fetal body weight, or incidences of fetal
malformations/variations. Thus, in the definitive study, the maternal toxicity NOAEL and LOAEL were 100 and 200 mg/kg/day,
respectively, and the developmental toxicity NOAEL was >200
mg/kg/day.
Key Words: melatonin; prenatal toxicity; development; mammary gland; pregnancy.
Melatonin (CAS No. 73–31– 4) is an endogenous substance
1
To whom correspondence should be addressed at MD B3-05 NIEHS, Bldg.
101 Alexander Drive, Research Triangle Park, NC 27709. Fax: (919) 541–
4634. E-mail: [email protected].
produced by the mammalian pineal gland and other organs,
especially the enterochromaffin cells of the gastrointestinal
tract and the retina (Hardeland et al., 1993; Huether, 1993).
Endogenous production of melatonin in humans has been estimated as 25–30 mg/day (Peters, 1992), or roughly 0.4 mg/kg/
day in a 70-kg person. Circulating levels of melatonin follow a
diurnal pattern, with night-time plasma levels higher than
day-time levels even in nocturnal species such as the rat
(Edmonds and Stetson, 1995; Reiter, 1991; Ronco and Halberg, 1996; Velàzquez et al., 1992). During pregnancy, the
diurnal pattern is maintained (Matsumoto et al., 1991;
Velàzquez et al., 1992), but peak plasma levels may vary with
stage of pregnancy (Kennaway et al., 1981; Pang et al., 1987).
Melatonin is a hydrophobic molecule that readily penetrates
biological membranes appearing in tissues or body fluids at
concentrations which are the same order of magnitude as those
in plasma (Hardeland et al., 1993; Menendez-Pelaez and Reiter, 1993).
Rat offspring prior to postnatal day (pnd) 10 do not have the
pineal enzymes to synthesize melatonin (Reppert and Klein,
1978). However, melatonin crosses the placenta and can be
transferred in maternal milk (McMillen and Nowak, 1988;
Reppert et al., 1979; Reppert and Klein, 1978). These and other
studies suggest that the dam serves as a natural source of
melatonin for rat offspring during gestation and early lactation.
Furthermore, melatonin mediates maternal-fetal transfer of information related to circadian phase and photoperiod (Williams
et al., 1991; Velàzquez et al., 1992).
Exogenously administered melatonin influences reproduction in laboratory rodents and other animals, particularly
species which are seasonal breeders (Asher et al., 1994;
Peters, 1992; Rivest et al., 1986). Notable effects of exogenous melatonin in adult female rats include disruption of
normal estrous cycles and reduced fertility, apparently due
to suppression of luteinizing hormone (LH) releasing hormone and, hence, suppression of ovulation (Rivest, 1987;
Vaughan et al., 1976; Ying and Greep, 1973; Walker et al.,
1982). The ability of exogenous melatonin to block the
pre-ovulatory LH surge in mammals has resulted in its
271
272
JAHNKE ET AL.
evaluation as an oral contraceptive in women (McElhinny et
al., 1996; Silman, 1993).
In immature female Wistar rats, melatonin (100 mg /day, sc)
delays sexual maturation, and this effect is dependent upon the
time of day for dosing, being most sensitive 9 –11 h after light
onset (Rivest et al., 1985). Immature male rats treated with
melatonin (100 mg/day, sc) for 15–20 days exhibited abnormal
progression of spermatogenesis, decreased ability of Leydig
cells to produce testosterone, decreased number of human
chorionic gonadotropin binding sites, and decreased serum LH
at the end of the juvenile period (Olivares et al., 1989). Male
rats appear to be most sensitive to effects on sexual maturation
when melatonin is administered in the afternoon, between
20 – 40 days of age, and these effects appear to be reversible
(Lang et al., 1984). Investigators evaluating the effects of
exogenous melatonin on sexual maturation have suggested that
this compound may play an intrinsic role in the timing of
puberty in mammals (Lang et al., 1984; Rivest et al., 1986). In
pregnant Balb-c mice, exogenous melatonin did not have adverse effects when administered daily throughout gestation
(200 mg /day, ip, equivalent to 5.7 mg/kg/day in a 35-g mouse),
3 h before the end of the light period (San Martin et al., 1995).
The authors reported no significant differences in dam body
weight or total numbers of fetuses, live fetuses, or abortions
through gd 18 (San Martin et al., 1995). However, mammary
gland development, measured according to DNA content and
concentration, was significantly inhibited. Subcutaneous implants containing melatonin also inhibited mammary development during pregnancy in the red deer, a short day-seasonal
breeder (Asher et al., 1994).
Because melatonin is produced endogenously and also occurs naturally in some foods, it can be sold as a dietary
supplement in the U.S. under the Dietary Supplement Health
and Education Act of 1994, without pre-market approval from
the FDA. Developmental and reproductive toxicity studies
under current regulatory guidelines were not found in the
published literature. Therefore, the current developmental toxicity studies were undertaken due to the widespread use of
melatonin by the general population as a nonprescription sleep
aid. In addition, the proposed use of melatonin as an antioxidant/anticancer agent, contraceptive, or antiaging agent can be
expected to increase the population exposed to supra-physiological doses. The screening study served as a dose-range
finder for the definitive study and also included examination of
maternal serum hormones and mammary gland development.
MATERIALS AND METHODS
Chemistry. The identity of melatonin (CAS No. 73–31– 4) [Triple Crown
America, Inc., Perkasie, PA (Lot No. tca6040568)] was confirmed by infrared
and proton nuclear magnetic resonance spectroscopy. Purity determinations by
gas chromatography with mass-spectroscopy detection or liquid chromatography with ultraviolet detection did not identify any impurities greater than 0.1%
of the major peak. The purity was therefore estimated as 100%, consistent with
the producer’s certification.
Animals and husbandry. Male (breeders) and female CDt rats (caesarean-originated, barrier-sustained, Crl:CDt (SD)BR VAF/ Plust outbred albino
rats, Charles River Laboratories, Inc., Raleigh, NC) were used in these studies.
Animals were individually identified by eartag. After a 10-day quarantine
period, individual breeding pairs were cohabited overnight. The morning on
which sperm were found in the vaginal lavage (Hafez, 1970) was designated
as gd 0. Confirmed-mated females were assigned to treatment groups by
stratified randomization (15/group in the screening study or 25/group in the
definitive study), so that mean body weight on gd 0 did not differ among
treatment groups in either study. Maternal body weights, for confirmed pregnant females used in these studies, ranged from 220 to 275 g on gd 0.
Confirmed-mated females were individually housed in solid-bottom polycarbonate cages with stainless steel wire lids (Laboratory Products, Rochelle Park,
NJ) and certified hardwood cage litter (Sani-Chipt, P.J. Murphy, Montville,
NJ). Certified rodent feed (Purina Certified Rodent Chowt, #5002; PMI, St.
Louis, MO) and tap water were available ad libitum throughout both studies.
Environmental conditions were monitored and controlled by computer (Barber-Colman Network 8000 System, Barber-Colman Co., Loves Park, IL).
Light cycles were maintained on 12:12 light:dark. Temperature and relative
humidity ranged from 21.6 –24.6°C, and 46.2– 65.2% in the screening study
and from 21.6 –23.7°C and 48.8 –59.2% in the definitive study.
Treatment. On the afternoons of gd 6 through 19, time-mated rats were
administered melatonin in 0.5% aqueous methylcellulose. Previous melatonin
studies have shown that afternoon dosing resulted in optimal alteration of
reproductive/endocrine endpoints (Lang et al., 1984; Rivest et al., 1985). Dose
groups were 0, 1, 10, 100, 150, or 200 mg/kg/day, po, in the screening study
and 0, 50, 100, or 200 mg/kg/day, po, in the definitive study. Administered
volume (5 ml/kg) was based upon body weight taken prior to daily dosing.
Dose formulations were prepared twice for each study (set 1 and set 2) and
were stored under refrigeration and used within the period of proven stability
(21 days). Aliquots were submitted for verification of melatonin concentration
by high performance, liquid chromatography (HPLC). In the screening study,
pre-dose aliquots were within 98.2–107% of their nominal concentrations, and
post-dose aliquots were within 94.8 –107% of nominal, except for one postdose sample from the lowest dose group, which was reported as 65.8% of
nominal concentration. In the definitive study, pre-dose aliquots were within
96.2–109.2% of their nominal concentrations, and post-dose aliquots were
within 93.3–105.4% of nominal, except for the highest concentration of set 1
(65.4%) and the lowest concentration of set 2 (115.8%).
Maternal evaluations. In the screening study and definitive study, body
weight (g) of confirmed-mated females was recorded on the mornings of gd 0
and 20, on the afternoons of gd 6 through 19, and immediately following
sacrifice on gd 20. Females were observed for clinical condition at least
once/day on gd 0 to 5 (prior to dosing). From gd 6 through 19, females were
observed for clinical condition and signs of pharmacological activity or toxicity at daily dosing, and generally at 30 – 60 min thereafter. On gd 20, females
were observed for clinical condition at weighing and again at scheduled
termination. Feed and water consumption was monitored during both studies,
with measurements on the mornings of gd 0 and 20 and on the afternoons of
gd 6, 9, 12, 15, 18, and 19.
On gd 20, time-mated females were sacrificed by CO 2 asphyxiation. The
body, liver, and gravid uterus of each time-mated female were weighed.
Thoracic and abdominal cavities were examined. Ovarian corpora lutea were
counted. Pregnancy status was confirmed by uterine examination. Uterine
contents were examined to determine the number of implantation sites, resorptions, dead fetuses, and live fetuses. Dead fetuses were counted, weighed, and
discarded. Uteri which presented no visible implantation sites were stained
with ammonium sulfide (10%) in order to visualize any implantation sites
which might have undergone very early resorption (Salewski, 1964).
In the screening study only, the left fourth mammary gland was removed and
fixed in 10% neutral buffered formalin, and embedded in paraffin. Tissue
sections at the level of the mammary lymph node were stained with hemotoxylin and eosin. Similar areas (2 3 3 mm) taken near the lymph node were
evaluated for percentage of fat-pad area occupied by glandular structure
273
DEVELOPMENTAL EVALUATION OF MELATONIN
(image analysis performed on a Macintosh computer using the public domain
NIH Image program (http://rsb.info.nih.gov/nih-image/). Immediately after
termination, blood was collected from the abdominal vena cava by syringe and
transferred to serum separation tubes (Vacuutainert). Samples were then
centrifuged at 4°C. Serum from each dam was aliquoted into snap-top
cryovials and frozen at approximately –20°C. Serum estradiol, progesterone
(Double Antibody Estradiol and Coat-A-Count Progesterone radioimmunoassay kits, respectively, Diagnostic Products Corp., Los Angeles, CA), prolactin,
and LH (Amersham Life Science, Arlington Heights, IL) concentrations were
determined by 125I radioimmunoassay techniques. Serum hormones were measured using a double-antibody procedure, except that a solid-phase assay was
employed for progesterone. All assays for serum hormone analyses were
completed on sera, which had been only thawed once.
Embryo/fetal evaluations, screening study. On gd 20, fetuses were dissected from the uterus and counted. All live fetuses were weighed, sexed
(externally), and examined for external morphological abnormalities, including
cleft palate. These fetuses were placed on a moist paper towel over ice prior to
decapitation (Blair, 1971; Lumb and Jones, 1973). Thus, fetuses were terminated by decapitation under hypothermic anesthesia (Tyl and Marr, 1997).
Embryo/fetal evaluations, definitive study. Live fetuses were dissected
from the uterus and immediately placed on a moist paper towel over a tray of
ice, a procedure which induces anesthesia by lowering the core body temperature below 25°C All live fetuses were counted, weighed, and examined for
external morphological abnormalities, including cleft palate. Approximately
50% of the fetuses were terminated by decapitation and the remaining ones by
evisceration.
Approximately 50% of the fetal carcasses were sexed and examined for
visceral morphological abnormalities using a fresh-tissue dissection method
(Staples, 1974; Stuckhardt and Poppe, 1984). The same fetal carcasses were
decapitated prior to dissection. Fetal heads were fixed and decalcified in
Bouin’s solution and subsequently examined using a free-hand sectioning
technique (Wilson, 1965). All fetal carcasses were eviscerated (and sex determined for those not scheduled for a full visceral morphological examination),
and the skeletons were macerated and stained with alcian blue/alizarin red-S
stain (Marr et al., 1988). Intact fetal skeletons (i.e., those fetuses that were not
decapitated) were examined for skeletal morphological abnormalities.
Statistics. The unit for statistical measurement was the pregnant female or
the litter. For each statistical comparison, the alpha level was 0.05, and the
significance was reported as p . 0.05 (not significant), p , 0.05, or p , 0.01.
Statistics, screening study. Nonparametric tests applied to continuous
variables included the Kruskal-Wallis one-way analysis of variance by ranks
for among-group differences and, if significant (p , 0.05), the Mann-Whitney
U test for pairwise comparisons to the vehicle control group (Siegel, 1956;
Winer, 1962). A one-tailed Mann-Whitney U test was used for all parameters,
except that maternal and fetal body weight parameters, and maternal feed and
water consumption, were examined in a two-tailed test (Siegel, 1956). Jonckheere’s test for k independent samples (Jonckheere, 1954) was used to identify
significant dose-response trends.
Nominal scale measures were analyzed by a Chi-square test for independence for differences among treatment groups (Snedecor and Cochran, 1967),
and by the Cochran-Armitage test for linear trend on proportions (Agresti,
1990; Armitage, 1955; Cochran, 1954). When Chi-square revealed significant
(p , 0.05) differences among groups, a one-tailed Fisher’s Exact probability
test, with appropriate adjustments for multiple comparisons, was used for
pairwise comparisons between each treatment group and the control group.
Statistics, definitive study. Quantitative, continuous data (e.g., maternal
body weights, fetal body weights, feed consumption, etc.) were compared
among treatment groups by parametric statistical tests whenever Bartlett’s test
for homogeneity of variance was not significant. When Bartlett’s test indicated
a lack of homogeneity (p , 0.001), nonparametric statistical tests were applied
(Winer, 1962). A one-tailed test (i.e., Dunnett’s Test) was used for serum
hormone and mammary gland-area pairwise comparisons.
Parametric statistical procedures were applied to selected measures from this
developmental toxicity study. General Linear Models (GLM) procedures
(SASt Institute, Inc., Cary, NC) were applied to the Analyses of Variance
(ANOVA) and the Tests for Linear Trend. Prior to GLM analysis, an arcsinesquare root transformation was performed on all litter-derived percentage data
(Snedecor and Cochran, 1967). For litter-derived percentage data, the ANOVA
was weighted according to litter size. When a significant (p , 0.05) main
effect for dose occurred, Dunnett’s Multiple Comparison Test (Dunnett, 1955;
1964) was used to compare each treatment group to the control group for that
measure. A one-tailed test (i.e., Dunnett’s test) was used for all pairwise
comparisons to the vehicle control group, except that a two-tailed test was used
for maternal body and organ weight parameters, maternal feed and water
consumption, fetal body weight, and percent males per litter. Nonparametric
tests, applied to continuous and nominal scale variables, were the same as
those described for the screening study.
RESULTS
Screening Study
Confirmed pregnancy rates were high (93–100%) for all
groups. No maternal deaths occurred in this study, and the
primary treatment-related clinical sign was “rooting” behavior,
which showed an increased incidence at $100 mg/kg/day.
Maternal food and water intake showed no consistent doserelated effects for the treatment period as a whole (data not
shown).
Prior to treatment (gd 0 – 6), maternal body weight and
weight gain did not differ among groups. Mean maternal body
weight at the high dose was never below 94% of the mean
control weight. Body weight gain during treatment (gd 6 to 20)
and during gestation (gd 0 to 20) as well as corrected gain
(weight gain during gestation minus gravid uterine weight)
each showed a significant decreasing trend across treatment
groups. Weight gains during the treatment period and gestational weight gains were each significantly reduced in the 150and 200-mg/kg/day groups, while corrected weight gain was
significantly reduced only at 200 mg/kg/day. No treatmentrelated effects were noted for gravid uterine weight or maternal
liver weight (absolute or relative, data not shown).
Area of the mammary glandular tissue and serum hormone
concentrations of 17b-estradiol, progesterone, prolactin, and
LH on gd 20 showed no treatment-related effect (Table 1).
There were no significant treatment-related effects on prenatal
growth, viability, or external morphology. Average live litter
size in melatonin-treated groups was 94 –102% of the control
mean, and average fetal body weight in melatonin-treated
groups was 97–101% of the control mean (data not shown).
Definitive Study
Maternal. Twenty-five time-mated female rats were assigned to each treatment group in this study, and pregnancy
was confirmed in all of these females (Table 2). Clinical signs
associated with melatonin exposure were minimal and no maternal deaths occurred in this study (Table 2).
Rooting in the cage bedding after dosing indicated an aversion to the taste, odor, or other sensory properties of the dose
274
JAHNKE ET AL.
TABLE 1
Glandular Area in the Mammary Fat Pad and Serum Hormone Concentrations of Term Pregnant Rats after
Melatonin Dosage on GD 6 –19
Melatonin (mg/kg/day, po)
Mammary gland area (%)
17b-Estradiol (pg/ml)
Luteinizing hormone (ng/ml)
Prolactin (ng/ml)
Progesterone (ng/ml)
0
1
10
100
150
200
59.5 6 2.7
9.5 6 1.1
1.8 6 0.2
30.7 6 5.0
35.0 6 3.8
59.5 6 2.6
13.5 6 2.5
1.8 6 0.1
27.2 6 4.4
29.8 6 3.0
57.5 6 2.5
11.1 6 1.9
1.8 6 0.1
43.0 6 11.5
32.9 6 3.3
58.7 6 3.7
14.8 6 2.7
1.7 6 0.1
24.2 6 4.6
31.8 6 2.4
52.9 6 1.8
14.2 6 3.5
1.8 6 0.1
16.6 6 2.6
26.5 6 5.1
60.0 6 2.1
9.1 6 1.8
1.8 6 0.1
37.1 6 5.9
28.2 6 3.6
Note. There were no statistically significant (p , 0.05) differences between the dosed groups and the control for any of these variables (Dunnett’s test).
Reported as the mean 6 SEM (N, 15 animals per group). Mammary glandular area in the fat pad of age-matched non-pregnant rats is ;26%.
formulation. On individual treatment days, rooting behavior
frequently showed a dose-related increase of incidence across
the 50-, 100-, and 200-mg/kg/day groups, but was never observed in the control group (data not shown). The peak incidence occurred on gd 15, when rooting was observed in 0, 16,
96, and 100% of the females from the control through highdose groups, respectively.
Maternal body weight did not differ among groups at any
time during the study, but weight gain was significantly decreased in the high-dose group during the first 3 days of
treatment (gd 6 to 9). This effect was less pronounced between
gd 9 and 12 (significant trend test only), and was not evident
during subsequent measurement periods (data not shown). Maternal weight gain during treatment (gd 6 to 20), during gestation (gd 0 to 20), and corrected weight gain did not differ
among groups (Table 2).
Prior to the initiation of treatment, relative maternal food
intake (g/kg/day) did not differ among groups. Between gd
6 and 12, maternal food intake was significantly decreased
at the high dose (Fig. 1). In contrast, the low-dose group (gd
12 to 18) and the mid-dose group (gd 15 to 18) exhibited
significantly greater food intake than controls, as did the
low-dose group for the treatment period as a whole (Fig. 1).
Maternal water intake was equivalent among groups prior to
TABLE 2
Maternal Toxicity in CDt Rats Exposed to Melatonin on Gestational Days 6 through 19
Melatonin (mg/kg/day)
Maternal pregnancy status
No. treated
No. (%) pregnant at sacrifice
Maternal body height (g) a
Gd 0 b
Gd 20 at sacrifice
Maternal body weight changes (g) a,b
Gestation wt gain (gd 0 to 20)
Treatment wt gain (gd 6 to 20)
Corrected wt gain c
Gravid uterine wt
Maternal organ weights a
Liver: Absolute (g)
Liver: Relative (% body wt) d
0
50
100
200
25
25 (100)
25
25 (100)
25
25 (100)
25
25 (100)
253.2 6 2.4
380.6 6 4.8
251.8 6 2.3
380.9 6 3.8
251.0 6 2.3
379.5 6 3.7
249.4 6 2.8
375.2 6 3.8
127.4 6 3.6
112.5 6 2.8
42.89 6 2.65
84.54 6 1.99
129.0 6 2.8
112.9 6 2.5
45.40 6 2.58
83.63 6 2.26
128.5 6 2.6
113.1 6 2.6
41.65 6 2.17
86.82 6 2.10
125.8 6 2.9
107.1 6 2.7
42.27 6 2.85
83.51 6 1.82
15.66 6 0.36
4.11 6 0.08‡
16.57 6 0.24
4.35 6 0.06*
16.23 6 0.24
4.28 6 0.06
16.55 6 0.29
4.41 6 0.06**
Includes all dams pregnant at sacrifice; mean 6 SEM; gd, gestational day.
Body weights were recorded in the morning of each designated gestational day.
c
Weight change during gestation minus gravid uterine weight.
d
Calculated using body weight at the time of sacrifice on gd 20.
‡p , 0.01; test for linear trend.
*p , 0.05; Dunnett’s test.
**p , 0.01; Dunnett’s test.
a
b
275
DEVELOPMENTAL EVALUATION OF MELATONIN
FIG. 1. Maternal gestational feed intake. Melatonin significantly decreased maternal feed intake for gd 6 to 9 and gd 9 to 12 at 200 mg/kg/day, and
significantly increased maternal feed intake for gd 12 to 15 at 50 mg/kg/day
and for gd 15 to 18 at 50 and 100 mg/kg/day. Data are presented as mean 6
SEM for 24 –25 pregnant dams per group. *p , 0.05 vs. the control group.
the initiation of treatment and throughout the treatment
period (data not shown).
Absolute maternal liver weight did not differ among groups
on gd 20, but relative liver weight (% body weight) was
increased in the low- and high-dose groups (Table 2). The
absence of a clear dose-response pattern suggests that this
apparent effect on relative liver weight may have been a
spurious result. Gravid uterine weight was equivalent among
groups (Table 2).
At termination, a limited number of gross findings primarily
relating to the urinary tract were reported for dams assigned to
this study (data not shown). Histopathology was not elected for
these maternal tissues, since individual findings failed to exhibit a dose-related incidence and were probably not related to
melatonin exposure. Furthermore, urinary tract anomalies are
common in this species and strain (Chandra and Frith, 1993/
1994; Humes et al., 1980; Van Winkle et al., 1988), and the
observed incidence may be affected by altered urodynamics
during pregnancy (Hsia and Shortliffe, 1995).
Developmental. Melatonin did not affect any of the endpoints related to embryo/fetal growth, viability, or morphological development (Tables 3 and 4). Average live-litter size in
melatonin-treated groups was between 98 –103% of the control
mean, and average fetal body weight per litter in melatonintreated groups was 100 –101% of the control mean (Table 3).
Likewise, the incidences of prenatal mortality (resorptions
and/or late fetal deaths), as well as fetal morphological anomalies (malformations or variations), were statistically equivalent among groups (Table 3).
DISCUSSION
Melatonin has been reported to influence the rate of reproductive maturation in rodents and may play an intrinsic role in
the timing of puberty in mammals. Furthermore, melatonin has
been reported to alter endocrine and reproductive status in
mature mammals and appears to act as an intrinsic modifier of
reproductive patterns in species sensitive to changes in photoperiod (Lang et al., 1984) The ability of exogenous melatonin
TABLE 3
Developmental Toxicity in CDt Rat Fetuses following Maternal Exposure to Melatonin on GD 6 through 19
Melatonin (mg/kg/day)
Number of litters a
No. corpora lutea/dam
No. implantation sites/dam
% preimplantation loss/litter
% resorptions/litter
% litters with resorptions
No. live fetuses/litter c
Avg. male fetal body wt/litter (g)
Avg. female fetal body wt/litter (g)
% male fetuses/litter
% externally malformed fetuses/litter d
% viscerally malformed fetuses/litter
% skeletally malformed fetuses/litter
% malformed fetuses/litter d
a
0
50
100
200
25
16.08 6 0.40 b
15.52 6 0.40
3.58 6 1.25
3.83 6 0.98
48
14.92 6 0.41
3.58 6 0.05
3.43 6 0.05
48.01 6 2.08
0.46 6 0.32
0.80 6 0.08
0
0.88 6 0.67
25
16.44 6 0.42
15.20 6 0.37
7.48 6 2.03
4.17 6 0.93
56
14.60 6 0.41
3.62 6 6 0.06
3.45 6 0.06
48.97 6 2.16
0
0.50 6 0.50
0.67 6 0.67
0.57 6 0.40
25
16.72 6 0.44
15.88 6 0.31
4.73 6 1.26
3.61 6 0.98
44
15.32 6 0.36
3.61 6 0.05
3.46 6 0.05
49.56 6 2.40
0
0.50 6 0.50
0
0.25 6 0.25
25
16.56 6 0.24
15.60 6 0.28
5.78 6 1.12
5.00 6 1.45
52
14.80 6 0.32
3.60 6 0.05
3.39 6 0.05
49.32 6 3.22
0
1.44 6 0.80
0
0.74 6 0.41
Includes all dams pregnant at sacrifice; litter size, no. implantation sites per dam.
Reported as the mean 6 SEM.
c
Every litter contained one or more live fetuses.
d
Fetuses with one or more malformations.
b
276
JAHNKE ET AL.
TABLE 4
Morphological Abnormalities in CD Rat Fetuses: Listing by Defect Type
Melatonin (mg/kg/day, po)
External Malformations a
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with external malformations c
% fetuses (% litters) with external malformations d
Anal atresia
Short thread-like tail
Short thread-like tail with bulbous tip
Visceral Malformations
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with visceral malformations c
% Fetuses (% litters) with visceral malformations d
Hydronephrosis: bilateral, left or right
Skeletal Malformations
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with skeletal salformations c
% Fetuses (% litters) with skeletal malformations
Thoracic centrum: Bipartite cartilage, bipartite ossification center e
External Variations
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with external variations c
% Fetuses (% litters) with external variations d
Visceral Variations
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with visceral variations c
% Fetuses (% litters) with visceral variations d
Enlarged lateral ventricle: bilateral, left, right
Distended ureter: bilateral, left or right
Skeletal Variations
No. fetuses (no. litters) examined b
No. fetuses (no. litters) with skeletal variations c
% Fetuses (% litters) with skeletal variations d
Misaligned sternebrae
Rudimentary rib, lumbar I: bilateral, left, or right
Short rib: XIII
Thoracic centrum:
Normal cartilage, bipartite ossification center
Dumbbell cartilage, dumbbell ossification center
Dumbbell cartilage, bipartite ossification center
0
50
100
200
373 (25)
2 (2)
0.5 (8)
2 (2)
1 (1)
1 (1)
365 (25)
0 (0)
0 (0)
383 (25)
0 (0)
0 (0)
370 (25)
0 (0)
0 (0)
187 (25)
2 (1)
1 (4)
2 (1)
182 (25)
1 (1)
0.5 (4)
1 (1)
193 (25)
1 (1)
0.5 (4)
1 (1)
185 (25)
3 (3)
2 (12)
3 (3)
186 (25)
0 (0)
0 (0)
183 (25)
1 (1)
0.5 (4)
1 (1)
190 (25)
0 (0)
0 (0)
185 (25)
0 (0)
0 (0)
373 (25)
0 (0)
0 (0)
365 (25)
0 (0)
0 (0)
383 (25)
0 (0)
0 (0)
370 (25)
0 (0)
0 (0)
187 (25)
39 (20)
21 (80)
35 (18)
6 (6)
182 (25)
36 (15)
20 (60)
31 (15)
6 (4)
193 (25)
31 (17)
16 (68)
21 (12)
15 (13)
185 (25)
33 (17)
18 (68)
26 (14)
10 (8)
186 (25)
10 (7)
5 (28)
1 (1)
1 (1)
183 (25)
20 (13)
11 (52)
190 (25)
17 (11)
9 (44)
185 (25)
17 (10)
9 (40)
3 (3)
2 (2)
1 (1)
3 (2)
5 (3)
1 (1)
3 (3)
12 (9)
6 (4)
1 (1)
8 (6)
4 (3)
3 (2)
7 (6)
3 (1)
2 (2)
a
In listing individual defects, a single fetus may be represented more than once, and data are presented as the number of fetuses (number of litters) exhibiting
that defect.
b
Only live fetuses were examined. The number of litters examined includes all litters with live fetuses.
c
Number of fetuses (number of litters) with one or more malformations/variations.
d
% Fetuses (% litters) with one or more malformations/variations. Percentages .1% are rounded to the nearest whole percent.
e
The centrum (including cartilaginous and ossified areas) was discontinuous across the midline.
to block the pre-ovulatory LH surge in mammals has resulted
in its evaluation as an oral contraceptive in humans (McElhinny et al., 1996; Silman, 1993). Thus, the endocrine activity
of melatonin is widely recognized.
Despite these effects on endocrine status and fertility, earlier
studies in laboratory rodents and other domestic species suggested that melatonin did not affect prenatal growth, survival,
or morphology of the conceptus once pregnancy had been
established (Chan and Ng, 1994, 1995; Tigchelaar and Nal-
bandov, 1975; Vaughan et al., 1976). The present investigations (NTP, 1997, 1998) extend the dose range evaluated in
pregnant animals, and the results are consistent with earlier
studies that failed to find adverse effects on prenatal growth,
viability, or gross morphological development. Furthermore,
these studies are the first to evaluate pregnancy outcome in
animals exposed repeatedly during gestation via the most common human route of administration.
Prenatally-induced effects on sexual maturation of the off-
277
DEVELOPMENTAL EVALUATION OF MELATONIN
spring are suggested by prior research in which gestational
exposure to melatonin (;2–2.5 mg/kg/day, sc) resulted in
delayed vaginal opening and/or altered LH levels postnatally
(Colmenero et al., 1991; Dı́az López et al., 1995; Vaughan et
al., 1970). Thus, studies designed to assess reproductive development and fertility in offspring exposed orally to melatonin in utero also appear to be warranted.
Melatonin administered during pregnancy inhibits mammary gland development (lobuloalveolization) in the mouse
(Sanchez-Barcelo et al., 1990; San Martı́n et al., 1995) and in
the red deer (Asher et al., 1994). Melatonin also inhibits
development of the mammary gland in organ culture (SanchezBarcelo et al, 1990), suggesting a direct inhibition of growth
during lobuloaveolar development. The increase in serum concentrations of estrogen, progesterone, prolactin, and glucocorticoids during pregnancy allows lobuloalveolization of the
mammary gland to occur (Dembinski and Shiu, 1987; Ichinose
and Nandi, 196; Vonderhaar 1987). In some studies demonstrating inhibition of mammary gland growth (Asher et al.,
1994), serum prolactin levels were also inhibited, suggesting
that inhibition of mammary gland growth by melatonin may be
mediated by lower prolactin levels.
In the current study, we found no effect on gd 20 of exogenously administered melatonin on the glandular area of the
mammary tissue, nor were there differences in estradiol, progesterone, prolactin, or LH serum concentrations when compared to control values. Lack of an effect of melatonin on LH
and FSH levels during pregnancy has been described in Holtzmann rats, although continuation of melatonin exposure suppressed the LH surge after delivery (Tigchelaar and Nalbandov, 1975).
Our findings are in apparent disagreement with the previous
study of melatonin inhibition of mouse mammary development
during pregnancy. Although the mouse and the rat have similar
reproductive and mammary developmental patterns, the response of mouse and rat mammary tissue to melatonin may be
different. The Balb/c mouse strain used in the experiments that
demonstrated inhibition is melatonin-deficient, lacking enzymes necessary for melatonin synthesis (Ebihara et al, 1987,
1986). Perhaps without endogenous melatonin, the biochemistry of the mammary growth factors or the melatonin receptor is
perturbed sufficiently by exogenous melatonin to result in a
measurable inhibition of growth. A similar argument can be
presented for inhibition of mammary gland growth by melatonin in tissue culture.
In this study, we used a different exposure period and
route of administration to the dams. In the mouse studies
and in deer, melatonin was given by a non-oral route
throughout the gestational period. Differences in experimental design, including route and duration of exposure, could
also account for the lack of effect on mammary gland
development in this study. The effect of these variables and
the role of the melatonin receptor on mammary growth
response are areas for further investigation.
In summary, no significant embryo/fetal toxicity was noted
in Sprague-Dawley-derived (CDt) rats dosed by gavage with
melatonin (0, 50, 100, or 200 mg/kg/day) from gd 6 through
19. Aversion to the dose formulations was noted at all doses, as
indicated by rooting in the cage bedding after daily gavage.
Mild maternal toxicity was noted at 200 mg/kg/day, based on
a transient reduction in body weight gain. Thus, the maternal
LOAEL in the definitive developmental toxicology study was
200 mg/kg/day, and the maternal NOAEL was 100 mg/kg/day.
The developmental NOAEL in this study was $200 mg/kg/
day.
Orally administered melatonin is associated with a wide
range of potential human exposures. Recommended doses are
reported as 0.2–10 mg for sleep induction, 1–10 mg for jet lag,
0.1–3 mg for anti-aging, 1–5 mg for shift work, or 2–20 mg for
immune stimulation (Reiter and Robinson, 1995). Melatonin
(75 mg) plus norethisterone (0.3 mg), administered over a
4-month period, resulted in anovulation, and this combination
is under clinical evaluation as an estrogen-free oral contraceptive (McElhinny et al., 1996; Reiter and Robinson, 1995;
Silman, 1993; Voordouw et al., 1992). Melatonin has also been
investigated as a component of cancer chemotherapy (10 mg/
day–50 mg 4 times/day; Conti and Maestroni, 1995; Robinson
et al., 1995) or in the management of hyperpigmentation disorders (250 mg, 4 times/day; Nordlund and Lerner, 1977).
Thus, the developmental toxicity NOAEL in this developmental toxicology study was ;1,400 –70,000 times the doses recommended for sleep induction; ;187 times the proposed daily
oral contraceptive dose; ;69 – 667 times the doses reported in
cancer patients; and ;14 times the reported dose in patients
with hyperpigmentation disorders.
ACKNOWLEDGMENTS
The laboratory animal studies were done under NIEHS Contract Number
N01-ES-65405 at Research Triangle Institute, Research Triangle Park, NC.
The laboratory studies were conducted under FDA-GLP guidelines, with the
exception of the determination of mammary gland area and serum hormone
levels in the screening study. Paraffin-embedded mammary tissue and frozen
serum were shipped from Research Triangle Institute and assayed at NIEHS.
Analytical chemistry support was provided under NIEHS Contract Number
N01-ES-55395 at Battelle Memorial Institute, Columbus, Ohio. Following
completion of the animal studies, the Sponsor provided for retention of records
under NIEHS Contract Number NO1-ES-35370 at Experimental Pathology
Laboratories, Inc., Research Triangle Park, NC. The authors would like to
thank the technical staff at Research Triangle Institute and the NIEHS chemistry support staff. Furthermore, we thank Dr. J. Haseman, NIEHS, for the
statistical analysis of Table 1 and Mr. Norris Flagler, NIEHS, for his help in
performing the image analysis of the mammary gland tissue.
REFERENCES
Agresti, A. (1990). Categorical Data Analysis. John Wiley and Sons, New
York.
Armitage, P. (1955). Test for linear trends in proportions and frequencies.
Biometrics 11, 375–386.
Asher, G. W., Veldhuizen, F. A., Morrow, C. J., and Duganzich, D. M. (1994).
278
JAHNKE ET AL.
Effects of exogenous melatonin on prolactin secretion, lactogenesis, and
reproductive seasonality of adult female red deer (Cervus elaphus). J.
Reprod. Fertil. 100, 11–19.
Blair, E. (1971). Hypothermia. In Textbook of Veterinary Anesthesia (L. Soma,
Ed.), pp. 555–579. Williams and Wilkins, Baltimore, MD.
Chan, W. Y., and Ng, T. B. (1994). Development of pre-implantation mouse
embryos under the influence of pineal indoles. J. Neural Transm. Gen. Sect.
96, 19 –29.
Chan, W. Y., and Ng, T. B. (1995). Changes induced by pineal indoles in
post-implantation mouse embryos. Gen. Pharmacol. 26, 1113–1118.
Chandra, M., and Frith, C. H. (1993/1994). Non-neoplastic renal lesions in
Sprague-Dawley and Fischer-344 rats. Exp. Toxicol. Pathol. 45, 439 – 447.
Cochran, W. (1954). Some methods for strengthening the common x 2 tests.
Biometrics 10, 417– 451.
Colmenero, M. D., Dı́az, B., Miguel, J. L., Gonzàlez, M. L. I., Esquifino, A.,
and Marı́n, B. (1991). Melatonin administration during pregnancy retards
sexual maturation of female offspring in the rat. J. Pineal Res. 11, 23–27.
Conti, A., and Maestroni, J. M. (1995). The clinical neuroimmunotherapeutic
role of melatonin in oncology. J. Pineal Res. 19, 103–110.
Dembinski, T. C. and Shiu R. P. (1987) Growth factors in mammary gland
development and function. In: The Mammary Gland: Development, Regulation, and Function (M. C. Neville and C. W. Daniel, Eds.), pp. 355–373.
Plenum Press, New York.
Dı́az López, B., Colmenero Urquijo, M. D., Diaz Rodriguez, M. E., Arce
Fraguas, A., Esquifino Parras, A., and Marı́n Fernàndez, B. (1995). Effect of
pinealectomy and melatonin treatment during pregnancy on the sexual
development of the female and male rat offspring. Eur. J. Endocrinol. 132,
765–770.
Dunnett, C. W. (1955). A multiple comparison procedure for comparing
several treatments with a control. J. Am. Stat. Assoc. 50, 1096 –1121.
Dunnett, C. W. (1964). New tables for multiple comparisons with a control.
Biometrics 20, 482– 491.
Ebihara, S., Hudson, D. J., Marks, T., and Menaker, M. (1987). Pineal indole
metabolism in the mouse. Brain Res. 416, 136 –140.
Ebihara, S., Marks, T., Hudson, D. J., Menaker, M. (1986). Genetic control of
melatonin synthesis in the pineal gland of mice. Science 231, 491– 493.
Edmonds, K. E., and Stetson, M. H. (1995). Pineal gland and melatonin affect
testicular status in the adult marsh rice rat (Oryzomys palustris). Gen. Comp.
Endocrinol. 99, 265–274.
Hafez, E. S. E. (Ed.) (1970). Reproduction and Breeding Techniques for
Laboratory Animals. Lea and Febiger, Philadelphia, PA.
thews and R. F Seamark, Eds.), pp. 123–136, Elsevier/North-Holland Biomedical Press, New York.
Lang, U., Aubert, M. L., Rivest, R. W., Vinas-Bradtke, J. C., and Sizonenko,
P. C. (1984). Daily afternoon administration of melatonin does not irreversibly inhibit sexual maturation in the male rat. Endocrinology 115, 2303–
2310.
Lumb, W. V., and Jones, E. W. (1973). Anesthesia in laboratory and zoo
animals. In Veterinary Anesthesia, pp. 452– 453. Lea and Febiger, Philadelphia.
Marr, M. C., Myers, C. B., George, J. D., and Price, C. J. (1988). Comparison
of single and double staining for evaluation of skeletal development: The
effects of ethylene glycol (EG) in CD rats. Teratology 37, 476.
Matsumoto, T., Hess, D. L., Kaushal, K. M., Valenzuela, G. J., Yellon, S. M.,
and Ducsay, C. A. (1991). Circadian myometrial and endocrine rhythms in
the pregnant rhesus macaque: Effects of constant light and timed melatonin
infusion. Am J. Obstet. Gynecol. 165, 1777–1784.
McElhinny, A. S., Davis, F. C., and Warner, C. M. (1996). The effect of
melatonin on cleavage rate of C57BL/6 and CBA/Ca preimplantation embryos cultured in vitro. J. Pineal Res. 21, 44 – 48.
McMillen, I. C., and Nowak, R. (1988). Maternal pinealectomy abolishes the
diurnal rhythm in plasma melatonin concentrations in the fetal sheep and
pregnant ewe during late gestation. J. Endocrinol. 120, 459 – 464.
Menendez-Pelaez, A., and Reiter, R. J. (1993). Distribution of melatonin in
mammalian tissues: The relative importance of nuclear versus cytosolic
localization. J. Pineal Res. 15, 59 – 69.
Nordlund, J. J., and Lerner, A. B. (1977). The effects of melatonin on skin
color and on the release of pituitary hormones. J. Clin. Endocrinol. Metab.
45, 768 –774.
NTP (1997). Developmental Toxicity Screen for Melatonin (CAS No.
73–31– 4) Administered by Gavage to Sprague-Dawley (CDt) Rats on
Gestational Days 6 through 19. NTP Study No. TRP-97– 001. NTP/
NIEHS Contract No. NO1-ES-65405. Final Study Report, June 25,
1997.
NTP (1998). Developmental Toxicity Evaluation of Melatonin (CAS No.
73–31– 4) Administered by Gavage to Sprague-Dawley (CDt) Rats on
Gestational Days 6 through 19. NTP Study No. TER-96 – 003. NTP/NIEHS
Contract No. N01-ES-65405. Final Study Report (NTIS Accession Number
PB98 –137300), March 31, 1998.
Olivares, A. N., Valladares, L. E., Bustos-Obregon, E., and Nunez, S. M.
(1989). Testicular function of sexually immature rats chronically treated
with melatonin. Arch. Biol. Med. Exp. 22, 387–393.
Hardeland, R., Reiter, R. J., Poeggeler, B., and Tan, D.-X. (1993). The
significance of the metabolism of the neurohormone melatonin: Antioxidative protection and formation of bioactive substances. Neurosci. Biobehav.
Rev. 17, 347–357.
Pang, S. F., Tang, P. L., Tang, G. W. K., Yam, A. W. C., and Ng, K. W.
(1987). Plasma levels of immunoreactive melatonin, estradiol, progesterone, follicle stimulating hormone, and beta-human chorionic gonadotropin during pregnancy and shortly after parturition in humans. J.
Pineal Res. 4, 21–31.
Hsia, T. Y., and Shortliffe, L. M. (1995). The effect of pregnancy on rat urinary
tract dynamics. J. Urol. 154(2 Pt 2), 684 – 689.
Peters, A. R. (1992). Endocrine manipulation—toxicological frontiers. J. Reprod. Fertil. 45(suppl.), 193–201.
Huether, G. (1993). The contribution of extrapineal sites of melatonin synthesis to circulating melatonin levels in higher vertebrates. Experientia 49,
665– 670.
Reiter, R. J. (1991). Pineal melatonin: Cell biology of its synthesis and of its
physiological interactions. Endocr. Rev. 12, 151–180.
Humes, H. D., Dieppa, R. A., and Brenner, B. M. (1980). Glomerular dynamics in rats with hereditary hydronephrosis. Invest. Urol. 18, 46 –51.
Reiter, R. J., and Robinson, J. (1995). A master sex hormone, and Taking
melatonin supplements. In Melatonin (R. J. Reiter and J. Robinson, Eds.),
pp. 158 –171 and 265–281. Bantam Books, New York.
Ichinose, R. R. and Nandi, S. (1966). Influence of hormones on lobulo-alveolar
differentiation of mouse mammary glands in vitro. J. Endocrinol. 35,
331–340.
Reppert, S. M., Chez, R. A., Anderson, A., and Klein, D. C. (1979). Maternalfetal transfer of melatonin in the nonhuman primate. Pediatr. Res. 13,
788 –791.
Jonckheere, A. R. (1954). A distribution-free k-sample test against ordered
alternatives. Biometrika 41, 133–145.
Reppert, S. M., and Klein, D. C. (1978). Transport of maternal [ 3H]-melatonin
to suckling rats and the fate of [ 3H]-melatonin in the neonatal rat. Endocrinology 102, 582–588.
Kennaway, D. J., Matthews, C. D., and Seamark, R. F. (1981). Pineal function
in pregnancy: Studies in sheep and man. In: Pineal Function (C. D. Mat-
Rivest, R. W. (1987). The female rat as a model describing patterns of pulsatile
DEVELOPMENTAL EVALUATION OF MELATONIN
LH secretion during puberty and their control by melatonin. Gynecol.
Endocrinol. 1, 279 –293.
Rivest, R. W., Aubert, M. L., Lang, U., and Sizonenko, P. C. (1986). Puberty
in the rat: modulation by melatonin and light. J. Neural. Transm. 21(suppl.),
81–108.
Rivest, R. W., Lang, U., Aubert, M. L., and Sizonenko, P. C. (1985). Daily
administration of melatonin delays rat vaginal opening and disrupts the first
estrous cycles: Evidence that these effects are synchronized by the onset of
light. Endocrinology 116, 779 –787.
Robinson, W. A., Dreiling, L., Gonzalez, R., and Balmer, C. (1995). Treatment
of human metastatic malignant melanoma with high dose oral melatonin. In
The Pineal Gland and its Hormones: Fundamentals and Clinical Perspectives (F. Fraschini, R. J. Reiter, and B. Stankov, Eds.), pp. 219 –225. Plenum
Press, New York.
Ronco, A. L., and Halberg, F. (1996). The pineal gland and cancer. Anticancer
Res. 16, 2033–2039.
Salewski, E. (1964). Farbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Naunyn-Schmiedebergs Arch. Exp.
Pathol. Pharmakol. 247, 367.
Sanchez-Barcelo, E. J., Mediavilla, M. D., and Tucker, H. A. (1990). Influence
of melatonin on the mammary gland growth: In vivo and in vitro studies.
Proc. Soc. Exp. Biol. Med. 194, 103–107.
San Martı́n, M., Recio, J., Sànchez-Barceló, E. J., and Mediavilla, M. D.
(1995). Melatonin inhibits mammary gland development during pregnancy.
Neuroendocrinol. Lett. 17, 245–250.
Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences.
McGraw-Hill, New York.
Silman, R. E. (1993). Melatonin: A contraceptive for the nineties. Eur. J.
Obstet. Gynecol. Reprod. Biol. 49, 3–9.
Snedecor, G. W., and Cochran, W. G. (1967). Statistical Methods, 6th Ed.
Iowa State University Press, Ames, Iowa.
Staples, R. E. (1974). Detection of visceral alterations in mammalian fetuses.
Teratology 9, 7.
Stuckhardt, J. L., and Poppe, S. M. (1984). Fresh visceral examination of rat
and rabbit fetuses used in teratogenicity testing. Teratog. Carcinog. Mutagen. 4, 181–188.
Tigchelaar, P. V., and Nalbandov, A. V. (1975). The effect of the pineal gland
on ovulation and pregnancy in the rat. Biol. Reprod. 13, 461– 469.
279
Tyl, R. W., and Marr, M. C. (1997). Developmental toxicity testing: Methodology. In Handbook of Developmental Toxicology (R. D. Hood, Ed.), pp.
175–225. CRC Press, New York.
Van Winkle, T. J., Womack, J. E., Barbo, W. D., and Davis, T. W. (1988).
Incidence of hydronephrosis among several production colonies of outbred
Sprague-Dawley rats. Lab. Anim. Sci. 38, 402– 406.
Vaughan, M. K., O’Steen, W. K., and Vaughan, G. M. (1970). Estrous cycle
changes after prenatal and neonatal injections of melatonin in normal and
androgen-sterilized female rats. Neuroendocrinology 6, 10 –18.
Vaughan, M. K., Reiter, R. J., and Vaughan, G. M. (1976). Fertility patterns in
female mice following treatment with arginine vasotocin or melatonin. Int.
J. Fert. 21, 65– 68.
Velàzquez, E., Esquifino, A. I., Zueco, J. A., Ruiz Albusac, J. M., and
Blazquez, E. (1992). Evidence that circadian variations of circulating melatonin levels in fetal and suckling rats are dependent on maternal melatonin
transfer. Neuroendocrinology 55, 321–326.
Vonderhaar, B.K. (1987). Prolactin: Transport, function, and receptors in
mammary gland development and differentiation. In The Mammary Gland:
Development, Regulation, and Function (M. C. Neville and C. W. Daniel,
Eds.) pp. 383– 417. Plenum Press, New York City.
Voordouw, B., Euser, R., Verdonk, R., Alberda, B., DeJong, F., Drogendijk,
A., Fauser, B., and Cohen, M. (1992) Melatonin and melatonin-progestin
combinations alter pituitary-ovarian function in women and can inhibit
ovulation. J. Clin. Endocrinol. Metab. 74, 108 –117.
Walker, R. F., McCamant, S., and Timiras, P. S. (1982). Melatonin and the
influence of the pineal gland on timing of the LH surge in rats. Neuroendocrinology 35, 37– 42.
Williams, L. M., Martinoli, M. G., Titchener, L. T., and Pelletier, G. (1991).
The ontogeny of central melatonin binding sites in the rat. Endocrinology
128, 2083–2090.
Wilson, J. G. (1965). Embryological considerations in teratology. In Teratology: Principals and Techniques (J. G. Wilson and J. Warkany, Eds.), pp.
251–277. University of Chicago Press, Chicago, IL.
Winer, B. J. (1962). Statistical Principles in Experimental Design. McGraw
Hill, New York.
Ying, S.-Y., and Greep, R. O. (1973). Inhibition of ovulation by melatonin in
the cyclic rat. Endocrinology 92, 333–335.