TOXICOLOG1CAL SCIENCES 4 1 , 8 8 - 9 9 (1998)
ARTICLE NO. T X 9 7 2 4 0 0
Pre- and Postnatal Toxicity of the HMG-CoA Reductase
Inhibitor Atorvastatin in Rats
J. W. Henck,* ' W. R. Craft,* A. Black.t J. Colgin,t and J. A. Anderson*
Departments of *Pathology and Experimental Toxicology, tPharmacokinettcs and Drug Metabolism, and ^Biometrics,
Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105
Received December 2, 1996; accepted October 22, 1997
Pre- and Postnatal Toxicity of the HMG-CoA Reductase Inhibitor Atorvastatin in Rats. Henck, J. W., Craft, W. R., Black, A.,
Colgin, J., and Anderson, J. A. (1998). Toxicol. Sci. 41, 88-99.
Atorvastatin is a potent inhibitor of the enzyme 3-hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate and constitutes
the rate-limiting step in the biosynthesis of cholesterol. Steroid
hormones derived from cholesterol, as well as mevalonate and
its isoprenoid derivatives, provide important contributions to the
maternal animal during pregnancy and lactation, as well as to the
growth and development of the offspring; these contributions may
potentially be influenced by inhibition of HMG-CoA reductase.
To investigate the effects of atorvastatin on various aspects of
reproduction and development, female Sprague-Dawley rats received 0, 20, 100, or 225 mg/kg daily by gavage from gestation
day 7 through lactation day 20. Maternal toxicity, characterized
by morbidity/mortality (13%), reduced body weight gain and food
consumption, and pathologic lesions in the nonglandular mucosa
of the stomach, occurred at 225 mg/kg. Offspring survival at birth
and during the neonatal period at 225 mg/kg was reduced relative
to control by up to 45%, and 28% of litters had no viable offspring
by 10 days postpartum. Additional effects on offspring included
reduced body weight during the neonatal and maturation periods
(100, 225 mg/kg), delayed appearance of pinnae detachment and
incisor eruption (225 mg/kg), impaired rotorod performance (females only; 100, 225 mg/kg), reduced acoustic startle responding
(males only; 20, 100, 225 mg/kg), and transient effects on shuttle
avoidance (females only; 225 mg/kg). No treatment-related effects
were observed on offspring reproduction. In a separate experiment,
a single dose of 10 mg/kg atorvastatin administered to female
Wistar rats on gestation day 19 or lactation day 13 provided evidence of placenta! transfer and excretion into the milk. Results
of this study indicate that pre- and postnatal administration of
atorvastatin to female rats produces developmental toxicity in
their offspring via in utero and/or lactational exposure, and in the
presence or absence of maternal toxicity. c i998Sodoyof Toxfco4o»y.
Atorvastatin is a potent inhibitor of the enzyme 3-hy1
To whom correspondence should be addressed at Parke-Davis Pharmaceutical Research, 2800 Plymouth Road, Ann Arbor. MI 48105. Fax: (313)
998-5718.
1096-6080/98 $25.00
Copyright C 1998 by the Society of Toxicology
All rights of reproduction in any form reserved
droxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase,
which catalyzes the conversion of HMG-CoA to mevalonate
and constitutes the rate-limiting step in the biosynthesis of
cholesterol (Fig. 1). By inhibiting HMG-CoA reductase activity in the liver, atorvastatin increases the cellular demand
for cholesterol, resulting in an increase in hepatic low-density lipoprotein (LDL) receptors and an increase in clearance
of plasma LDL by the liver (Kovanen et al., 1981). Since
most cholesterol is transported in LDL in humans, decreased
plasma concentrations of LDL cholesterol would be anticipated in patients with hyperlipidemia. Several inhibitors of
HMG-CoA reductase (vastatins) are currently being used
clinically in the treatment of hyperlipidemia; atorvastatin
has been shown to produce even greater reductions in LDL
cholesterol in hyperlipidemic patients than those produced
by vastatins currently in use (Nawrocki et al., 1995).
Inhibition of HMG-CoA reductase results in reduced levels of cholesterol, a precursor in the biosynthesis of hormones important in the maintenance of pregnancy, parturition, lactation, and maternal behavior (Numan, 1988; Freeman, 1988; Hodgen and Itskovitz, 1988). In addition,
cholesterol is required for normal development in mammals
both in utero and during the postnatal period (Belknap and
Dietschy, 1988; Yount and McNamara, 1991). HMG-CoA
reductase inhibition also results in decreased concentrations
of mevalonate and its many isoprenoid derivatives, including
dolichol, isopentenyl adenine, ubiquinone, haem A, and
many other growth-regulating proteins bound to farnesyl and
geranylgeranyl residues (Soma et al., 1992; Brewer et al.,
1993; Corsini et al., 1995) (Fig. 1). These isoprenoids are
involved in membrane synthesis, DNA replication, cellular
growth and metabolism, and protein glycosylation, processes
crucial to normal development (Quesney-Huneeus et al.,
1983; Surani et al., 1983; Farnsworth et al., 1987; Brewer
et al., 1993).
The majority of vastatins are developmentally toxic in rats.
Lovastatin was developmentally toxic in rats when administered during organogenesis, producing a treatment-related
increase in the incidence of the malformation gastroschisis
(Minsker et al, 1983). Pre- and postnatal administration of
89
PRE- AND POSTNATAL TOXICITY OF ATORVASTATIN
acetyl-CoA + acetoacetyl-CoA
3-hydroxy-3-methylglutaryl-CoA [HMG-CoA]
,
HMG-CoA reductase
mevalonate
mevalonate - PP
dimethylallyl - PP
isopentenyl - PP
geranyl - PP
proteins
dolichols
isoprenoic acids
ubiquinones
geranylgeranyl - PP
isopentenyl adenine (tRNA)
E
famesyl - PP
proteins
haem A
isoprenoic acids
squalene
cholesterol •
—
—
—
—
steroid hormones
vitamin D
bile acids
hpoproteins
FIG. 1. The biosynthetic pathway which yields mevalonate, isoprenoids, and cholesterol. The enzyme HMG-CoA reductase catalyzes the rate-limiting
step in this pathway; inhibition of this enzyme by agents such as vastatins results in reduced levels of mevalonate and subsequent products. Adapted
from Goldstein and Brown (1990), Soma el al. (1992), and Corsini et al. (1995).
lovastatin to rats resulted in decreased postweaning survival
and delayed development of reflexes and behavior (FDA,
1987). Simvastatin was not teratogenic, but resulted in reduced fetal body weight when given to female rats during
organogenesis (Wise et al., 1990a), reduced fetal and offspring body weights when given prior to mating and throughout gestation (Wise et al., 1990b), and reduced offspring
body weight and increased swim maze errors when given
perinatally (Minsker et al., 1990). Fluvastatin resulted in
reduced offspring body weight and decreased neonatal survival when administered to rats from late gestation throughout lactation (Hrab et al., 1994), and was developmental^
toxic, as evidenced by retarded skeletal development and
reduced fetal body weights, when administered prior to mating and throughout gestation and lactation (FDA, 1993).
No adverse effects on reproduction, fertility, or survival
of the conceptus occurred following oral atorvastatin administration to male or female rats prior to and during mating
(Dostal et al., 1996). However, oral administration of ator-
vastatin to pregnant rats and rabbits during the period of
organogenesis resulted in developmental toxicity, characterized by increased postimplantation loss and decreased fetal
body weight, but not teratogenicity, at maternally toxic doses
(Dostal et al., 1994). The present study was conducted to
further characterize findings of developmental toxicity by
extending the treatment period from organogenesis through
delivery and weaning of the offspring. This exposure period
was designed to evaluate maternal effects during pregnancy
and lactation (including effects on nursing and maternal
care), as well as effects on delivered offspring, including
survival, growth, development, behavior, and reproductive
performance.
MATERIALS AND METHODS
Pre- and Postnatal Toxicity
Animals. Sexually mature male and female Sprague-Dawley rats
(Crl:CD BR VAF/Plus; Charles River Breeding Laboratories, Inc., Portage,
90
HENCK ET AL.
• Ca(
R G . 2.
Chemical structure of atorvastatin.
MI) were used to evaluate the pre- and postnatal toxicity of atorvastatin.
On gestation day (GD) 0, females were 12 to 13 weeks of age and weighed
226 to 314 g, at the time mating was initiated, untreated males were 12 to
13 weeks of age and weighed 340 to 401 g. Following an acclimation
period of at least 1 week, animals with no adverse clinical signs were
assigned to the study. Each animal received unique identification and was
housed individually (except during mating and lactation) in a stainless steel,
hanging wire mesh cage. Near the time of parturition, a solid stainless steel
plate and bedding were added to the home cage of each female. Food
(Purina Certified Lab Rodent Chow No. 5002, Ralston Purina Co., St. Louis,
MO) and water were available ad lib throughout the study. Environmental
conditions were in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH Publication 86-23, 1985). Untreated males and
females were paired in a 1.1 ratio until mating was confirmed by the
presence of sperm in the vaginal smear (GD 0). The females were then
randomly assigned to treatment groups of 30 animals each. Of these, 25
contributed litters which were evaluated through adulthood, while 5 served
as satellite animals from which plasma was collected for pharmacokinetic
determinations.
Test material
Atorvastatin is described chemically as [/?-{fl*,/?*)]-2(4-fluorophenyl)-/9,<5-dihydroxy-5-(l-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1 W-pyrrole-1 -heptanoic acid calcium salt (2:1) (Fig. 2). It
was supplied as a white amorphous powder by the Clinical Pharmaceutical
Operations Department of the Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company (Ann Arbor, MI), and had a stated active
moiety of 91.2%. Dosing aliquots were prepared by suspending bulk atorvastatin in aqueous 0.5% methylcellulose (MC) at specific concentrations
based on the active moiety content. Samples from each concentration of
dosing suspension were analyzed periodically for drug concentration during
the treatment period; results were within 10% of the intended value.
Treatment.
Fo generation female rats received 0 (0.5% MC vehicle),
20, 100, or 225 mg/kg atorvastatin daily by oral gavage from GD 7 through
Lactation Day (LD) 20 at a dose factor of 10 mL/kg body wt. Dose volumes
were based on the most recent individual body weights, with the exception
that dose volumes continued to be based on the GD 15 body weight until
the LD 0 body weight was obtained. The treatment period was selected to
cover maternal and developmental events occurring during embryonic and
fetal development, parturition, and lactation. F, generation offspring were
exposed to atorvastatin in utero and postnatally during lactation. The oral
route of administration was selected because it is the intended clinical route
for atorvastatin. Doses for this study, with an anticipated dosing period of
approximately 5 weeks, were selected based on results of a 2-week oral
dose range-finding study and a 13-week oral toxicity study in nonpregnant
female rats (unpublished data), as well as the teratology study in rats
previously cited (Dostal el ai, 1994). No mortality occurred at 250 mg/kg
in the 2-week study, but five females were euthanized moribund at 225
mg/kg in the 13-week study, and one female died at 300 mg/kg in the
teratology study. No-adverse-effect doses in these studies were 70, 20, and
100 mg/kg, respectively. The greatest emphasis was placed on the teratology study; increased postimplantation loss at 300 mg/kg indicated that this
dose was too high to achieve a sufficient number of live litters in the
present study Therefore, the high dose of 225 mg/kg in the 13-week study
was adopted. The high dose of 225 mg/kg was expected to cause some
maternal toxicity without excessive mortality, while the low dose of 20
mg/kg was expected to represent a no-adverse-effect dose, as recommended
in ICH guidelines (1994).
F, generation. Fo generation animals were monitored for clinical observations, body weight, and food consumption. Each dam was handled
daily during gestation in an attempt to minimize maternal stress and any
ensuing effects on offspring during the postnatal period. All Fo females were
allowed to deliver and rear their offspring. Each Fo female was euthanized
following weaning or death of her litter, and a gross necropsy examination
was conducted.
F, generation. F, generation offspring were evaluated for survival,
clinical observations, growth, development, behavior, and reproduction. On
postnatal day (PND) 4, each litter was culled to eight offspring (four/sex,
whenever possible), and on PND 21, two/sex/litter were euthanized for
gross necropsy examination. The PND of acquisition of the following
developmental landmarks was noted in all surviving offspring: pinnae detachment, lower incisor eruption, eye opening, testes descent, and vaginal
opening.
Behavior tests conducted in postweaning and/or mature offspring (one/
sex/litter) included rotorod, acoustic startle, motor activity, and two-way
active avoidance. Rotorod performance (Omni-Rotor Treadmill, Omnitech
Electronics, Inc., Columbus, OH) was evaluated on PND 28. Each animal
received three training trials and three test trials at a constant rotor speed
of 10 rpm, with a maximum duration of 60 s/trial. Mean time on the rotorod
was utilized for group mean comparison. Locomotor activity was evaluated
in an automated activity monitor (Digiscan Animal Activity Monitor, Omnitech Electronics, Inc., Columbus, OH) on PND 42 during one 4-min test
session, designed to evaluate initial response to the novel environment of
the activity monitor, as well as to observe the rate of habituation to that
environment over the four consecutive 1 -min test periods. The 4-min length
of the test session has been demonstrated in our laboratory to identify
changes in the rate of response to a novel open field environment (Henck
el ai, 1995, 1996). The acoustic startle response, as well as prepulse inhibition and habituation to an acoustic startle stimulus, was evaluated on PND
43. Each animal was given 70 trials using the SR-LAB Startle Response
System (San Diego Instruments, San Diego, CA); the trials alternated between a background noise of 70 dB (total of 30 trials), a noise level tone
of 120 dB (total of 20 trials), and a prepulse tone of 90 dB followed in
100 ms by a noise level tone of 120 dB (total of 20 trials), separated by
intertrial intervals ranging from 5 to 30 s. Peak response for the first 120dB trial, the rate of habituation over 20 120-dB trials, and percentage
91
PRE- AND POSTNATAL TOXICITY OF ATORVASTATIN
response inhibition resulting from the prepulse tones were evaluated. On 4
consecutive days during PN week 9, animals were tested for acquisition of
a learned behavior (Learning Phase) and, after a 3-day rest period, for
memory retention (Recall Phase) using an active avoidance paradigm in a
two-compartment shuttle box (Shuttle Scan, Omnitech Electronics, Columbus, OH). Animals were tested for 25 trials on each testing day The conditioned stimulus was light and the unconditioned stimulus was footshock
with a duration of 10 s. Footshock levels were 0.33 mA for males and 0.22
mA for females; different levels were required due to the sexually dimorphic
response to footshock in rats (Beany and Beatty, 1970; Demi and Epstein,
1972). Parameters which were evaluated included percentage avoidance
response, percentage escape response, and mean shock duration (the amount
of time, up to 10 s/trial, for which footshock was endured prior to an
avoidance or an escape)
Sexually mature offspring (one/sex/litter, excluding animals tested for
behavior) were evaluated for reproductive potential by 1:1 pairings of nonlittermates from the same treatment group. Mated F, females underwent
cesarean section on GD 21, maternal and fetal parameters (including examination for external abnormalities) were evaluated. Following behavior testing or evaluation of reproductive potential, surviving F, offspring were
euthanized and subjected to gross necropsy evaluation.
Pharmacokinelics
On LD 8 of the pre- and postnatal study, venous blood was obtained
under anesthesia from five Fo control females 4 h postdose and from three
to five Fo females/atorvastatin treatment group at 0 (predosing), 2, 4. 8, 12,
and 24 h postdose, for plasma drug concentration analysis. LD 13 was
selected as a time during which the volume of milk produced by the rat is
adequate for analytical procedures (Fiorotto el ai. 1991).
In a separate set of experiments, sexually mature female Wistar rats
[Crl:(WI)BR VAF/Plus, Charles River Breeding Laboratories, Inc., Kingston, NY] were used to evaluate the placental transfer and milk excretion
of radiolabeled atorvastatin. Although the strain of rat was different from
that used to evaluate pre- and postnatal toxicity, the answers obtained in
Wistar rats regarding the existence of placental transfer and milk excretion
in the rat as a species were considered applicable to Sprague-Dawley rats.
To evaluate placental transfer, six rats were given a single dose of 10
mg/kg of a [l4C]atorvastatin suspension in 0.5% methylcellulose (average
radioactive dose 62 JJCX) by oral gavage on GD 19. Animals were anesthetized and hepannized blood samples were collected by cardiac puncture 6
h postdose; whole blood was centrifuged, and the resultant plasma was
analyzed by liquid scintillation spectrometry for radioactivity content. The
6-h timepoint was selected based on time to maximum atorvastatin plasma
concentrations in a bioavailability study in rats (unpublished data). Following blood collection, animals were euthanized, and the following tissues
were obtained for analysis of radioactivity content by liquid scintillation
spectrometry following combustion- maternal liver, placenta, whole fetuses
(pooled samples from 1/2 of the fetuses/each litter), and fetal liver (pooled
samples from the remaining 1/2 of the fetuses/each litter).
To determine whether atorvastatin was excreted into rat milk, a second
set of six female Wistar rats was given a single dose of 10 mg/kg of a
['4C]atorvastatin suspension in 0.5% methylcellulose (average radioactive
dose 48 fid) by oral gavage on LD 13. Each female's litter was removed
approximately 3 h postdose to facilitate milk letdown. Approximately 5
min prior to milk collection at 6 h postdosing, each female was anesthetized
and primed with approximately 1.5 1U of oxytocin. Approximately 1.5
mL of milk was collected over a maximum 30-min interval. Immediately
following milking, heparinized blood samples were collected by cardiac
puncture; whole blood was centrifuged, and the resultant plasma was analyzed for radioactivity content. Following blood collection, animals were
euthanized, and their livers were harvested for analysis of radioactivity
content, as indicated previously. An additional three female rats and their
nursing offspring were anesthetized 6 h postdose on LD 13, and hepannized
blood samples were collected by cardiac puncture for subsequent analysis
of maternal and offspring plasma radioactivity content. Following blood
collection, animals were euthanized, and maternal and offspring livers were
harvested for analysis of radioactivity content.
Statistical Analysis
To control for the multiplicity of statistical comparisons (i.e., reduce the
number of false positive conclusions), all sets of maternal, reproductive,
developmental, and behavioral response measures (parameters) were divided into distinct classes based on the relationship of the parameters. For
example, all maternal body weight measurements comprised one class,
while all developmental landmark data comprised another. The level of
significance for each comparison within the class was 0.05 divided by the
square root of the number of parameters in the class (Tukey el ai, 1985),
to provide an approximate classwise significance level of 5%.
The basic trend test employed was the sequential trend test, using the
rank dose scale and rank-transformed data (Park, 1985; Tukey el ai. 1985).
This test is equivalent to sequential application of a Kruskal—Wallis oneway analysis of variance by ranks (Kruskal and Wallis, 1952), with the
treatment effects being evaluated by dose-trend tests which have contrast
coefficients for equally spaced (ranked) treatment groups. The sequential
linear trend test is designed to detect monotone changes in dose response:
it does not detect trend reversal or curvature. If the true dose response was
not monotonic, the trend test was considered not sensitive enough to detect
the treatment effect A trend reversal test was then conducted, consisting
of a quadratic trend test performed at the two-tailed I % classwise significance level. If the trend reversal test was significant and the high-dose trend
test was not significant, the treatment groups were compared to the control
by Dunnett's test (Dunnett, 1955, 1964), using rank-transformed data, and
performed at the 5% classwise significance level
The monotonic dose-response relationship tested by the trend test was
not considered realistic for the activity monitor parameters because they
have been demonstrated with several drugs to exhibit a U-shaped doseresponse curve (Iversen and Iversen, 1981). Hence, Dunnett's test on ranktransformed data was performed in place of the trend test as the main
analytical method. In addition to trend analysis, acoustic startle data were
subjected to profile analysis (Johnson and Wichem, 1982) to evaluate the
acoustic startle response by treatment group interaction (parallelism test)
and, secondarily, to address the question of equal group effects; the raw
data were used for this analysis. The methods of Greenhouse and Geisser
(1959) were incorporated in the profile analysis to produce conservative
tests for the parallelism hypothesis.
RESULTS
Fo Generation
Maternal toxicity, characterized by mortality and reduced
body weight and food consumption, occurred at 225 mg/kg
(Table 1). Two animals in this group died, one animal was
euthanized in moribund condition, and one animal was euthanized due to inability to deliver. Ten dams at 225 mg/kg
were euthanized by LD 10 because they had no viable offspring remaining. The most common treatment-related gross
pathologic finding in dams at 225 mg/kg found dead or
euthanized prior to scheduled termination was abnormal surface of the nonglandular mucosa of the stomach. Body
weight gain at 225 mg/kg was significantly (p < 0.0189)
less than control by 14% during the gestation treatment period, but was significantly (p < 0.0189) greater than control
92
HENCK ET AL.
TABLE 1
Maternal Parameters in Dams Treated with Atorvastatin from GD 7 through LD 20
Dose (mgAg)
Parameter
0
20
100
225
No. sperm-positive females
No. died/euthanized prior to
scheduled termination
Body weight change (g)c
GD7-21
LD 0-21
Food consumption (g/animal)r
GD7-2I
LD0-2I
Gestation duration (days)c
Litter size (livebom)c
Postimplantation loss (%)r
No. euthanized because no viable
offspring remained
30
30
30
30
2°
(23)
(24)
128 ± 4 4
17.3 ± 3.09
(23) 352 ± 6.3
(22) 1125 ± 14.9
(24) 21 9 ± 0.08
15.0 ± 0.44
(24)
(24)
9.0 ± 1.39
(22)
(22)
130 ± 4.7
18.5 ± 4.05
(22) 360
(22) 1088
(22) 22.2
14.7
(22)
(22)
10.6
0
0
4
1"
0
± 8.3
±343
± 0.13
± 0.81
± 1.86
(23)
(23)
135 ± 3.5
30.3 ± 3.84
(23) 362
(23) 1067
(24) 22.0
(24)
15.0
(24)
10.2
0
± 5.8
± 18.7
± 0.07
± 0.62
± 2.33
(20) 100 ± 4.9*
(8) 36.4 ± 9.70*
(20) 325
(7) 866
(18) 21.9
(18) 14.1
(18) 11 3
± 6.6*
± 79.5*
± 0.08
± 0.67
± 2.41
10
Note. GD, gestation day; LD, lactation day.
° Deaths attributed to gavage error.
b
Euthanized due to prolapsed uterus.
c
(N) Mean ± standard error; treatment groups compared statistically to vehicle control.
* p < 0.0189 (0 05 divided by the square root of seven individual parameters in the maternal body weight change class), different from vehicle control
for trend test.
by twofold during the lactation treatment period. Food consumption at 225 mg/kg was significantly {p < 0.0189) less
than control by 8% during the gestation treatment period and
by 23% during the lactation treatment period. No significant
differences from control occurred for duration of gestation,
litter size, or postimplantation loss at 225 mg/kg. No maternal parameters were affected by atorvastatin treatment at 20
or 100 mg/kg.
F, Generation
Survival at birth and on PND 4 and 21 was significantly
{p < 0.0289) less than control at 225 mg/kg by 5, 36, and
45%, respectively (Table 2). The majority of deaths in this
group occurred between PND 0 (including stillbirths) and
10, and were not preceded by clinical signs of morbidity.
Survival during the maturation period (PND 21-91) was
also significantly (p < 0.05) less than control for females
only, although only two females in this group died, as compared to no deaths in the control group.
Group mean body weights of male and female offspring
(Table 2) were significantly (p < 0.0289) less than control
at 100 mg/kg by 10-15% on PND 4 and 21, and at 225 mg/
kg they were significantly (p < 0.0289) less by 9-37% on
PND 0, 4, and 21. Group mean body weight gain during
PND 4 - 2 1 was also significantly (p < 0.0289) less than
control for males and females at 100 and 225 mg/kg, by
15-16 and 32-40%, respectively. While body weights at
100 mg/kg were essentially comparable to control by PN
days 42 and 70 for males and females, respectively, body
weights at 225 mg/kg continued to be less than control, and
were significantly (p < 0.0289) less for PN day 91 by 6
and 8% for males and females, respectively.
Pinnae detachment and eye opening were significantly
(p < 0.0224) delayed at 225 mg/kg relative to control by
approximately 1 and 1.5 days, respectively (Table 2). The
incidence of neonatal offspring (=s21 days of age) with the
anatomical variation dilated renal pelvis was increased at
225 mg/kg relative to control and other atorvastatin groups.
The incidence of dilated renal pelvis was comparable between control and atorvastatin groups after PND 21.
Rotorod performance of females at 100 and 225 mg/kg
was significantly (p < 0.05) less than control by 37 and
53%, respectively (Fig. 3). No treatment-related effects were
apparent on rotorod performance for males or on motor activity for males and females.
For acoustic startle testing, although not statistically significant by trend analysis, group mean maximum input voltage for Noise Level (120 dB) Trial 1 for males at 20, 100,
and 225 mg/kg was suppressed relative to control in a nondose-related manner by 48, 32, and 59%, respectively (Table
3). In addition, maximum input voltage for Noise Level Trial
2 for males at 225 mg/kg was suppressed by 50% (data
not shown). This reduced response to an acoustic stimulus
appeared to be transient, however, as responding in subse-
93
PRE- AND POSTNATAL TOXICITY OF ATORVASTATIN
TABLE 2
Physical Parameters in Offspring from Dams Treated with Atorvastatin from GD 7 through LD 20
Dose (mg/kg)
0
20
(24) 98.7 ± 0.73
(24) 98.7 ± 0.54
(24) 100
(22) 97.0 ± 2.36
(22) 98.4 ± 0.58
(22) 98.3 ± 1 25
(24) 95.8 ± 1.80
(23) 96.4 ± 1.28
(23) 100 ± 0
(24) 95 8 ± 2.88
(24) 100
(21) 100
(22) 100
(23) 100
(23) 100
(24)
(24)
6.4 ± 0.09
6.1 ± 0.08
(22)
(22)
6.6 ± 0.12
6.3 ± 0.12
(24)
(24)
6.4 ± 0.12
6.0 ± 0.08
(18)
(18)
5.8 ± 0.19*
5.4 ± 0.16*
(24)
(24)
10.2 ± 0.21
9.6 ± 0.20
(21)
(22)
10.4 ± 0.29
9.6 ± 0.27
(23)
(23)
9.2 ± 0.32*
8.6 ± 0.30*
(12)
(14)
7.2 ± 0.66*
6.4 ± 0.52*
Parameter
Survival (%)**
PN day 0
PN days 0 - 4 (preculling)
PN days 4-21
PN days 21-91
Males
Females
Body weight (g)°
PN day 0*
Males
Females
PN day 4*
Males
Females
PNday 21*
Males
Females
PN day 42
Males
Females
PN day 70
Males
Females
PN day 91"
Males
Females
Developmental landmarks"*
Pinnae detachment
Lower incisor eruption
Eye opening
Testes descent
Vaginal opening
Incidence of dilated renal pelvis in
neonates ( s PN day 21)
100
225
(18) 94.0 ± 1 77*
(18) 63.3 ± 10.3*
(14) 55.4 ± 13.3*
(7) 100
(8) 87.5 ± 12.5*
(24) 57.4 ± 0.98
(24) 54.5 ± 0.91
(21) 58.0 ± 1.70
(22) 53 5 ± 1.71
(23) 49.4 ± 1.70*
(23) 46.4 ± 1 58*
(7) 40.5 ± 4.57*
(8) 34.2 ± 4.31*
(24) 229
(24) 176
± 4.2
± 2.2
(21) 235
(22) 177
± 3.9
± 3.6
(23) 219
(23) 165
± 5.7
±28
(7) 198
(7) 146
± 12.3
± 7.5
(24) 438
(24) 267
± 6.7
±3.4
(21) 447
(22) 269
± 6.0
±50
(23) 424
(23) 259
±7.7
± 4.1
(7) 402
(7) 246
± 16.8
± 9.2
(24) 525
(24) 306
± 8.4
± 4.6
(21) 538
(22) 305
± 7.1
± 5.8
(23) 511
(23) 294
±8.5
± 4.3
(7) 493
(7) 280
± 16.9*
± 13.6*
(24) 2.9 ±
(24) 11.5 ±
(24) 14.5 ±
(24) 21.3 ±
(24) 32.7 ±
0.10
0.15
0.16
0.38
0.49
(22) 2.5 ±
(22) 10.7 ±
(22) 14.5 ±
(21) 20.9 ±
(22) 31 6 ±
0.9%
3.3%
0.15
0.21
0.19
0.36
0.26
(23) 2.8 ±
(23) 11.9 ±
(23) 15.2 ±
(23) 20.8 ±
(23) 33.4 ±
0.8%
0.10
0.14
0.19
0.31
0.89
(14) 3.7 ±
(8) 11.6 ±
(8) 16.1 ±
(7) 22.2 ±
(7) 34.1 ±
0.29**
0.45
0.50**
0.92
1 11
6.1%
Note. GD, gestation day; LD, lactation day; PN, postnatal.
° (Litter N) Mean ± standard error, sexes combined unless indicated otherwise.
'Treatment groups compared statistically to vehicle control.
• p < 0.0289 (0.05 divided by the square root of seven individual parameters in the maturation body weight class), different from vehicle control
for trend test.
** p < 0.0224 (0.05 divided by the square root of five individual parameters in the developmental landmarks class), different from vehicle control
for trend test.
quent trials was in general comparable to that of controls
(data not shown). No treatment-related effects were apparent
for Noise Level trials for females, and statistical comparison
across 20 Noise Level trials revealed no treatment-related
difference in the rate of habituation to repeated acoustic
stimuli for males or females. The degree of response inhibition following a prepulse stimulus for Prepulse Trial 1 (defined as the percentage response inhibition relative to the
preceding Noise Level trial) is also given in Table 4. No
treatment-related effect on response inhibition was evident
for males. However, response inhibition of all atorvastatin
treatment groups of females was greater than that of controls
for Trial 1, and, when all 20 trials were considered, group
mean response inhibition across all trials was 68, 86, 80,
and 84% for the controls and at 20, 100, and 225 mg/kg,
respectively (data not shown).
All shuttle avoidance parameters for males were comparable between atorvastatin groups and controls for both the
Learning and Recall phases of testing. Although not statistically significant, group mean shock duration for females at
94
HENCK ET AL.
FEMALES
o
10-
100
20
225
100
225
ma/ka Atorvastatin
ma/ka Atorvastatin
FIG. 3. Group mean and standard error rotorod performance in offspring from dams administered atorvastatin daily by gavage from gestation day 7
through lactation day 20. Each animal received three trials, with a maximum duration of 60 s/trial. The mean of these three trials was used to calculate
the group mean. 'Significantly different from control mean by trend test, p < 0.0500.
225 mg/kg was 68% and twofold greater than control on
test days 3 and 4 of the Learning Phase, respectively (Fig.
4). In addition, group mean percentage avoidance on test
day 4 for females at 225 mg/kg was 65% less than control.
All other shuttle avoidance parameters for females of all
atorvastatin groups were comparable to control for both the
Learning and Recall Phases of testing.
No treatment-related effects were apparent on F, generation reproductive parameters, including copulation and fertility indices, number of corpora lutea, litter size, and pre- and
postimplantation losses, or on F2 fetal parameters, including
body and placental weights, sex ratio, and external appearance (Table 4).
Pharmacokinetics
Maternal plasma pharmacokinetic parameters obtained in
the pre- and postnatal study on LD 8 are given in Table 5.
Mean plasma Cmsx and AUC(0-24) values increased with
increasing atorvastatin dose; this increase was more than
proportional to dose.
Pharmacokinetic data from the placental transfer and milk
excretion experiments are given in Table 6. I4C Radioactivity
was detected in fetal rats and fetal livers 6 h following oral
administration of [14C]atorvastatin to female rats on GD 19,
indicating that atorvastatin and/or its metabolites undergo
placental transfer. Mean fetal liver concentration was 0.176
fig eq/g, while mean concentration of the entire fetus was
TABLE 3
Group Mean Acoustic Startle Parameters for the First of 20 Noise Level and 20 Prepulse Trials, Including Percentage Response
Inhibition, in Offspring from Dams Treated with Atorvastatin from GD 7 through LD 20
Atorvastatin
(mg/kg)
0
0
20
20
100
100
225
225
Sex
Males
Females
Males
Females
Males
Females
Males
Females
Maximum input voltage.
Noise Level Trial 1'
(24)
(23)
(20)
(20)
(22)
(23)
(6)
(6)
973 ±
1106 it
502 ii
664 it
665 i:
1163 d:
3 % i:
716 ±
148.9
191.0
120.7
121.8
88.2
154.3
114.0
148.3
Maximum input voltage,
Prepulse Trial 1"
(24) 315
(24) 406
(20) 232
(21) 190
(22) 257
(23)318
(6) 146
(6) 77
±
±
±
±
±
±
±
±
78.2
66.4
69.9
55.5
70.8
68.1
64.1
12.6*
' (N) Mean ± standard error, first Noise Level (120 dB) and Prepulse (90 dB followed in 100 ms by 120-dB tone) trials.
* Percentage inhibition in response relative to Noise Level Trial 1.
* p < 0.0500, different from vehicle control for trend test.
% Response
inhibition*
68
63
54
71
61
73
63
89
95
PRE- AND POSTNATAL TOXICITY OF ATORVASTATIN
TABLE 4
Reproductive Parameters in Offspring from Dams Treated with Atorvastatin from GD 7 through LD 20
Dose (mg/kg)
Parameter
0
No. pairs cohabited
Copulation index (%)a*
Fertility index (%f'
Corpora lutea'
Litter size'
Preimplantalion loss*'
Postimplantation loss (%)dJ
F2 Fetal parameters
Body weight (g), live fetuses''
Males
Females
Sex ratio (%Y
Males
Females
Placenta weight (g)'
(18)
(20)
(18)
(24)
23
91 3
95.2
17.4 ±
13.2 ±
13.0 ±
9.0 ±
20
0.62
1.06
3.69
1.39
(17)
(17)
(17)
(22)
225
100
21
95.2
85.0
17.9 ±
15.2 ±
6.2 ±
10.6 ±
0.63
0.53
2.16
1.86
(19) 5.4 ± 0.08
(19) 5 1 ± 0.08
(17) 5.4
(17) 5.0
(19) 47.4 ± 2.84
(19) 52.6 ± 2.84
(19) 0.47 ± 0.015
(17) 46.9 ± 2.17
(17) 53.1 ± 2.17
(17) 0.4S ± 0.015
± 0.09
± 0.07
(20)
(20)
(20)
(24)
23
91.3
95.2
18.1 ±
15.3 ±
9.6 ±
10.2 ±
0.64
0.64
2.67
2.33
(6)
(6)
(6)
(18)
7
100
85.7
16.0 ±
12.0 ±
10.0 ±
113 ±
0.86
1.65
6.82
2.41
(20) 5.4 ± 0 0 6
(20) 5.1 ± 0.07
(6) 5.4
(6) 5.1
± 0.12
± 0.04
(20) 48.8 ± 2.92
(20) 51.2 ± 2 92
(20) 0.48 ± 0.008
(6) 43.4 ± 3.85
(6) 56.6 ± 3.85
(6) 0.54 ± 0.038
Note. All treatment group means are statistically comparable to control.
" (Number females with positive mating divided by number cohabited) x 100.
* Group mean comparisons conducted.
"(Number pregnant females divided by number with positive mating) X 100.
J
(JV) Mean ± standard error, treatment groups compared statistically to vehicle control.
' [(Number of corpora lutea — implant sitesVcorpora lutea] X 100.
^[(Number of implant sites — viable fetuses)/implant sites] X 100.
0.035 fig eq/g, suggesting hepatic extraction of radioactivity.
Pregnant rats also had high liver radioactivity concentrations,
with a mean of 26.7 fig eq/g, relative to mean placental
and plasma values of 0.111 fig eq/g and 0.178 fig eq/mL,
respectively.
I4
C Radioactivity was detected in nursing pups 6 h following oral administration of [ I4C]atorvastatin to lactating rats
on LD 13, indicating that atorvastatin equivalents undergo
milk excretion in lactating rats. Lactating rats whose pups
were removed 3 h prior to milk collection had mean plasma
and milk values of 0.099 and 0.102 fig eq/mL, respectively,
for a milk:plasma ratio of 1.56. The mean liver concentration
for these females was 8.22 fig eq/g. Nursing pups that remained with the dam through 6 h postdose had mean plasma
and liver concentrations of 0.050 fig eq/mL and 0.037 fig
eq/g. respectively. Dams of these pups had mean plasma and
liver concentrations of 0.077 fig eq/mL and 10.7 fig eq/g,
respectively.
DISCUSSION
Maternal toxicity, characterized by mortality, suppression
of body weight gain and food intake, and pathologic lesions,
occurred at 225 mg/kg. The plasma AUC(0-24) achieved
at this dose was 22 times the human AUC at the highest
recommended therapeutic dose of 80 mg/kg (Parke-Davis,
1996). Lesions observed in the nonglandular mucosa of the
stomach of females at 225 mg/kg were similar to those described in lactating rats treated with lovastatin (MacDonald
etal, 1988;Minskerefa/., 1990) or fluvastatin (FDA, 1993;
Hrab et al., 1994). Stomach lesions observed with fluvastatin
were shown to be specific to rodents and were considered
due to contact irritation (Robison et al., 1994). Maternal
body weight gain was less than that of controls at 225 mg/
kg during the gestation treatment period, but was actually
greater during lactation, a pattern similar to that observed
with lovastatin (FDA, 1987). It is unclear in the present
study why weight gain increased during lactation when food
consumption was suppressed to an even greater extent than
it was during gestation. Maternal body weight gain was also
greater than control at 100 mg/kg during lactation, but was
comparable to control during gestation; no treatment-related
effects on food consumption occurred at this dose. The biological significance of the dose-related increase in maternal
body weight gain during lactation cannot be ascertained, but
is not considered to represent maternal toxicity. Hrab and
co-workers (1994) determined that mevalonic acid supplementation of perinatally administered fluvastatin prevented
maternal toxicity, mortality, and cardiac myopathy; in addition, MacDonald and co-workers (1988) found that coadministration of mevalonic acid prevented lovastatin-induced lesions in the gastric mucosa. Hrab and co-workers (1994)
96
HENCK ET AL.
MEAN SHOCK DURATION
PERCENT AVOIDANCE RESPONSE
40
7 6 -
•
A
V
Vehicle control
20 mg/kg
100 mg/kg
•
225 mg/kg
•
35
A
V
30
•
Vehicle control
20 mg/kg
100 mg/kg
225 mg/kg
(0
0)
r
T
o
25 (0
'5
< 20
2 4
3 -
15
2 -
10
i
i
2
4
Day of Learning Phase
//\
/// ^
^
L
1
\
[
"I
Day of Learning Phase
FIG. 4. Group mean and standard error shuttle avoidance parameters during the Learning Phase in female offspring from dams administered atorvastatin
daily by gavage from gestation day 7 through lactation day 20. Data are mean shock duration for 25 trials/animal, with a maximum duration of 10 s/
trial, and percent of 25 trials which elicited an avoidance response Although not statistically significant when tested by trend analysis for each day of
the Learning Phase, group mean shock duration at 225 mg/kg was 68% and twofold greater than control on days 3 and 4 of the Learning Phase,
respectively, and percent avoidance responding was 65% less than control on day 4.
concluded that the adverse maternal effects observed with
fluvastatin were due to exaggerated pharmacologic activity
resulting from inhibition of the conversion of HMG-CoA to
mevalonate. Because atorvastatin employs the same mechanism of action as fluvastatin, it is likely that inhibition of
HMG-CoA reductase was also responsible for atorvastatininduced maternal toxicity.
No treatment-related malformations were observed in
pups stillborn or found dead on PND 0, consistent with the
lack of malformations in the teratology study in rats with
atorvastatin (Dostal et al., 1994); in contrast, although postimplantation loss was increased in the teratology study at
the highest dose of 300 mg/kg, no treatment-related effects
occurred on fetal survival in the present study. However,
survival at birth and throughout the preweaning period was
significantly reduced at the maternally toxic dose of 225 mg/
kg, and all offspring died in 28% of the litters. Treatment
of female rats during late gestation and throughout lactation
with lovastatin (FDA, 1987) or fluvastatin (FDA, 1993; Hrab
et al., 1994) also resulted in reduced offspring survival during the preweaning period at maternally toxic doses. This
increase in mortality was reversed by supplementation of
fluvastatin with mevalonic acid (Hrab et al., 1994), implicating inhibition of HMG-CoA reductase as a possible mechanism of action. The window of susceptibility to mortality
resulting from atorvastatin exposure was immediately prior
to birth (resulting in stillbirths) through the early postnatal
period. When the events occurred which resulted in stillbirths and early postnatal death is unknown. Pharmacokinetic data indicate that atorvastatin can be transferred from
the dam to the fetus during late gestation, and can also be
excreted into the milk during lactation, providing two routes
by which atorvastatin could have contributed to neonatal
mortality. However, the contribution of the dam and the
TABLE 5
Mean Plasma Pharmacokinetic Parameters Obtained on LD 8
from Dams Treated with Atorvastatin Beginning on GD 7°
Dose
(mg/kg)
(ng eq/mL)
(h)
AUQO-24)
(ng eq • h/mL)
20
100
225
104 (57.8)
1050 (85.4)
5780 (98.4)
2.4 (37.3)
3.2 (83.8)
2.0 (0.0)
803 (38.6)
6130(83.7)
21600(77.3)
Note C D . maximum observed plasma atorvastatin concentration; f™,,
time to reach C , ; AUQO-24), area under the plasma concentration-time
curve from time 0 to 24 h postdose.
" Data presented as mean (% relative standard deviation, RSD); N = 3
(225 mg/kg) or 5 (20 and 100 mg/kg).
PRE- AND POSTNATAL TOXICITY OF ATORVASTATTN
TABLE 6
Placental Transfer and Milk Secretion of Atorvastatin Equivalent Concentration 6 H after a Single 10 mg/kg Oral Dose of
[uC]Atorvastatin"
Pregnant and fetal rats {N = 6 litters)
Maternal
Fetal
Liver
Plasma
Placenta*
Fetus*
Liver*
26.7 (15)
0 178(18)
0 111(12)
0.035(26)
0.176(16)
Lactating rats (N = 6 litters)
Maternal
Liver
Plasma
Milk
8.22 (47)
0 099 (62)
0.102 (25) [ 1.56f
Lactating rats and pups (N = 3 litters)
Maternal
Liver
10.7 (7)
Plasma
0.077 (9)
Neonatal
Plasma*
0.050 (41)
Liver*
0.037 (92)
" Data presented as mean (% RSD) \i% eq/g or p.% eq/mL.
* Pooled samples.
' fMean plasma:milk ratio].
impact of maternal toxicity at this dose must also be considered. It is of interest that in a study which included perinatal
administration of fluvastatin alone or fluvastatin supplemented with mevalonic acid, there was a higher incidence
of dead pups without milk in the stomach among groups
administered fluvastatin alone (Hrab et ai, 1994), indicating
that pups did not suckle and/or dams did not nurse the pups.
Offspring body weights were reduced relative to those of
controls from birth through some portion of the maturation
period at 100 and 225 mg/kg, indicating that growth suppression occurred at these doses. Pinnae detachment and eye
opening were delayed at 225 mg/kg; these indications of
developmental delay are not unexpected, since achievement
of preweaning landmarks is highly correlated with body
weight (ICH, 1994). The incidence of dilated renal pelvis
was increased in offspring at 225 mg/kg; while this is not
an uncommon finding in rodent developmental toxicity studies, it is unclear if it represents a developmental delay or
a biologically significant anomaly (Kavlock et ai, 1988).
Developmental delay appears to be a likely scenario in this
study, however, because dilated renal pelvis was no longer
observed after PND 21.
Various effects on behavior were detected in offspring
from atorvastatin-treated groups. Rotorod performance of
females at 100 and 225 mg/kg was significantly impaired.
97
Although impairment may have been due to the reduced
body weights of these animals, rotorod performance in males
was unaffected in the presence of similar reductions in body
weight gain. The acoustic startle response in males of all
atorvastatin groups was suppressed relative to control during
the first one or two trials only of a multiple trial test session.
Although the initial response to a noise level tone was unaffected in females of all atorvastatin groups, the maximum
response generated during the first prepulse trial at 225 mg/
kg was less than that of controls; in contrast, response inhibition resulting from a prepulse stimulus was elevated for the
first trial and most remaining trials. It is unclear why response inhibition would be increased at 225 mg/kg; a deficit
in hearing would be expected to decrease response inhibition
because the ability to detect a prepulse tone would be impaired (Wecker et ai, 1985), while a deficit in motor function would be expected to reduce the response to the initial
noise level tone (Fechter and Young, 1983). Prepulse inhibition was unaffected in males and females, indicating that
atorvastatin was not ototoxic under the testing conditions,
but rather may have affected reactivity to initial acoustic
stimuli. Transient effects on shuttle avoidance (increased
mean shock duration and decreased percent avoidance) occurred during the learning phase in females only at 225 mg/
kg; no effects on retention of the learned response (memory)
were observed in males or females. Transient effects on
behavior were also observed with simvastatin; in a swim
maze test of learning and memory, female offspring from
dams treated with simvastatin during organogenesis had
more errors than controls at 7 to 8 weeks of age, but not at
10 to 11 weeks of age (Wise et ai, 1990b). Behavioral
effects in offspring from dams given lovastatin prior to mating and during gestation included delays in development of
the righting reflex, negative geotaxis, auditory startle, and
swimming ontogeny, as well as a reduction in the mean
latency period for open field (females of all dose groups)
(FDA, 1987); offspring from dams given lovastatin during
late gestation and lactation displayed a reduction in the
acoustic startle response, decreased responsiveness to the
free-fall righting reflex, and a reduced number of fecal pellets in the open field (FDA, 1987). Behavioral changes
which occurred at 100 and 225 mg/kg in the present study
may have been related to growth suppression and developmental delay, although a direct effect on the central nervous
system cannot be entirely ruled out.
Developmental toxicity induced by HMG-CoA reductase
inhibitors is prevented in vitro or in vivo by mevalonic acid,
but not by cholesterol or a specific inhibitor of cholesterol
synthesis (Minsker et al., 1983; Surani et al., 1983; Carson
and Lennarz, 1979; Hrab et al., 1994). Results of these studies indicate that products of the mevalonate pathway other
than cholesterol could be involved in the mechanism of action governing developmental toxicity. HMG-CoA reductase
98
HENCK ET AL.
activity demonstrates a specific developmental pattern: a
sharp rise in activity occurs immediately prior to birth (which
may coincide with the increased sterol synthesis required
for rapid tissue hypertrophy), followed by an equally sharp
postnatal decline and low levels of activity throughout most
of the suckling period; activity levels rise again following
weaning (McNamara et ai, 1972; Yount and McNamara,
1991). Pharmacokinetic data in the present study indicated
not only that atorvastatin was transferred across the placenta
on gestation day 19, but also that concentrations were higher
in the fetal liver than in the whole fetus. Thus, the potential
existed for atorvastatin to directly influence the late gestational increase in fetal hepatic cholesterol synthesis required
for cell growth and proliferation, synthesis and maintenance
of membranes, and synthesis of hormones and bile acids
(Quesney-Huneeus et ai, 1983; Belknap and Dietschy,
1988). The influence of HMG-CoA reductase inhibitors on
isoprenoid derivatives of mevalonate has not been characterized during late gestation in the rat; however, given the
important role these compounds play in membrane biogenesis, DNA replication, cellular growth and metabolism, and
protein glycosylation (Quesney-Huneeus et ai, 1983; Surani
et ai, 1983; Farnsworth et aL, 1987; Brewer et ai, 1993),
the potential exists for reductase inhibitors to alter development by reducing levels of isoprenoids.
Alteration of maternal levels of steroids synthesized from
cholesterol could potentially play a role in developmental
toxicity resulting from pre- and postnatal exposure to HMGCoA reductase inhibitors, although that role has not yet been
elucidated. Lactation is the most important means by which
neonates obtain cholesterol; therefore, hormonal disruption
of lactation could adversely affect neonatal survival and development. Because the activity of HMG-CoA reductase and
hepatic sterol synthesis is already low in suckling rat fetuses,
it is unlikely that reductase inhibition by atorvastatin during
this period would produce adverse developmental effects
directly, despite the fact that direct exposure occurs via the
milk. It is more likely that if developmental toxicity results
from events which occur after parturition, those events are
related to the maternal animal and may involve such factors
as the quantity and quality of milk and maternal behavior,
including nursing.
Results from this study indicate that maternal toxicity
occurred at 225 mg/kg, and that developmental toxicity,
characterized by increased preweaning mortality, reduced
body weight, developmental delay, and behavioral changes,
was of the greatest magnitude at this dose. Reduced offspring
body weight and behavioral changes also occurred at 100
mg/kg, a dose which did not result in adverse effects on
the maternal animal. Although diminished acoustic startle
responding occurred in male offspring at 20 mg/kg, the effect
was transient, and no other apparent treatment-related effects
occurred in dams or offspring at this dose. Therefore, 20
mg/kg was considered a no-adverse-effect dose for developmental toxicity under the conditions of this study, while 100
mg/kg was considered a no-adverse-effect dose for maternal
toxicity.
ACKNOWLEDGMENTS
The authors thank Debra Sherman and Roxann Alonzo for technical
assistance, and Y. Y. Shum for plasma drug concentration analyses.
REFERENCES
Beatty, W. W., and Beatty, P A. (1970) Hormonal determinants of sex
differences in avoidance behavior and reactivity to electric shock in the
rat. J. Comp. Physwl. Psychol. 3, 446-455
Belknap, W M., and Dietschy, J. M (1988). Sterol synthesis and low density lipoprotein clearance in vivo in the pregnant rat, placenta, and fetus.
J. Clin. Invest. 82, 2077-2085.
Brewer, L. M., Sheardown, S. A., and Brown, N. A. (1993). HMG-CoA
reductase mRNA in the post-implantation rat embryo studied by in situ
hybridization Teratology 47, 137-146.
Carson, D. D., and Lennarz, W. J. (1979). Inhibition of polyisoprenoid and
glycoprotein biosynthesis causes abnormal embryonic development.
Proc. Natl. Acad. Set. USA 76, 5709-5713.
Corsini, A., Maggi, F. M., and Catapano, A. L. (1995). Pharmacology of
competitive inhibitors of HMG-CoA reductase. Pharmacol. Res. 31, 9 27.
Denti, A , and Epstein, A. (1972). Sex differences in the acquisition of two
kinds of avoidance behavior in rats. Physiol. Behav. 8, 61 1-615.
Dostal, L. A., Schardein, J. L., and Anderson, J. A. (1994). Developmental
toxicity of the HMG-CoA reductase inhibitor, atorvastatin, in rats and
rabbits. Teratology 50, 387-394.
Dostal, L A., Whitfield, L. R , and Anderson, J. A. (1996). Fertility and
general reproduction studies in rats with the HMG-CoA reductase inhibitor, atorvastatin. Fundam. Appl. Toxicol 32, 285-292.
Dunnett, C. W (1955). A multiple comparison procedure for comparing
several treatments with a control. J. Am. Slat. Assoc. 50, 1096-1121.
Dunnett, C. W. (1964). New tables for multiple comparisons with a control.
Biometrics 20, 482-491.
Farnsworth, W. H., Hoeg, J. M., Maher, M., Brittain, E. H., Sherins, R. J..
and Brewer, H. B., Jr. (1987). Testicular function in Type II hyperlipoproteinemic patients treated with lovastatin (mevinolin) or neomycin. J. Cltn.
Endocrinol. Metab. 65, 546-550.
FDA (1987). Summary basis of approval of NDA 19-643 (lovastatin), 128.
FDA (1993). Summary basis of approval of NDA 20-261 (fluvastatin), 51 54, and Amendment, pp. 1 and 2.
Fechter, L. D., and Young, J. S. (1983). Discrimination of auditory from
nonauditory toxicity by reflex modulation audiometry: Effects of triethyltin. Toxicol. Appl. Pharmacol. 70, 216-227.
Fiorotto, M.L., Burrin, D. G., Perez, M., and Reeds, P. J (1991). Intake
and use of milk nutrients by rat pups suckled in small, medium, or large
litters. Am. J. Physiol. 260, Rl 104-RI113
Freeman, M. E (1988). The ovarian cycle of the rat. In The Physiology of
Reproduction, Vol. 2 (E. Knobil and J. D. Neill. Eds), pp. 1893-1928
Raven. New York.
Goldstein. J. L. and Brown. M S. (1990) Regulation of the mevalonate
pathway Nature 343, 425-430.
PRE- AND POSTNATAL TOXICITY OF ATORVASTATIN
Greenhouse, S. W., and Geisser, S. (1959). On methods in the analysis of
profile data. Psychometrika 32, 95-112.
Henck, J. W., Petrere, J. A., and Anderson, J. A. (1995). Developmental
neurotoxicity of CI-943, a novel antipsychotic. Neuroloxicol. Teratol. 17,
13-24.
Henck, J. W., Frahm, D. T., and Anderson, J. A. (1996). Validation of
automated behavioral test systems. Neuroloxicol. Teratol. 18, 189—197.
Hodgen, G. D., and Itskovitz, J. (1988). Recognition and maintenance of
pregnancy. In The Physiology of Reproduction, Vol. 2 (E. Knobil and
J. D. Neill, Eds.), pp. 1995-2021. Raven, New York.
Hrab, R. V., Hartmen, H. A., and Cox, R. H., Jr. (1994). Prevention of
fluvastatin-induced toxjcity, mortality, and cardiac myopathy in pregnant
rats by mevalonic acid supplementation. Teratology 50, 19-26.
ICH (1994). International Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. Fed. Reg. 59,
48746-48752.
Iversen, S. D., and Iversen, L. L. (1981). Behavioral Pharmacology. Oxford
University Press, New York
Johnson, R. A., and Wichern, D. W. (1982). Applied Multivariate Statistical
Analysis. Prentice-Hall, New York.
Kavlock, R. J , Hoyle, B. R., Rehnberg, B. F , and Rogers, E. H. (1988).
The significance of the dilated renal pelvis in the rutrogen-exposed rat
fetus: Effects on morphology and function. Toxicol. Appl. Pharmacol.
94, 287-296
Kovanen, P. T., Bilheimer, D. W., Goldstein, J. L , Jaramillo, J. J., and
Brown, M. S. (1981). Regulatory role for hepatic low density lipoprotein
receptors in vivo in the dog. Proc. Natl. Acad. Sci. USA 78, 1194-1198.
Kruskal, W. H., and Wallis, W. A. (1952). Use of ranks in one-criterion
variance analysis. J. Am. Stat Assoc. 47, 583-621.
MacDonald, J. S., Gerson, R. J., Kombrust, D. J., Kloss, M. W., Prahalda,
S., Berry, P. H., Alberts, A. W., and Bokelman, D. L. (1988). Preclinical
evaluation of lovastatin. Am. J. Cardiol. 62, 16J-27J.
McNamara, D. J., Quackenbush, F. W., and Rodwell, V. W (1972). Regulation of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A reductase. J.
Biol. Chem. 247, 5805-5810.
Minsker, D. H , MacDonald, J. S., Robertson, R. T., and Bokelman, D. L.
(1983). Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, and inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Teratology 28, 449-456.
99
Minsker, D. H., Robertson, R. T, Bokelman, D. L., Akutsu, S., and Fujii,
T. (1990). Simvastatin (MK-0733): Oral late gestation and early lactation
study in rats. Oyo Yahiri 39, 169-179.
Nawrocki, J W., Weiss, S. R., Davidson, M. H., Sprecher, D. L., Schwartz,
S. L., Lupien, P.-J., Jones, P. H., Haber, H. E., and Black, D. M. (1995).
Reductions of LDL cholesterol by 25% to 60% in patients with primary
hypercholesterolemia by atorvastatin, a new HMG-CoA reductase inhibitor. Arterioscler. Thromb. Vase. Biol. 15, 678-682.
Numan, M. (1988). Maternal behavior. In The Physiology of Reproduction,
Vol. 2 (E Knobil and J. D. Neill, Eds.), pp. 1569-1645. Raven, New
York.
Park, Y. C. (1985). Nonparametric sequential trend test. Proceedings of the
Tenth Annual SAS Users Group International Conference, pp. 809-813.
Parke-Davis, Division of Warner-Lambert Company (1996). Lipitor (atorvastatin calcium) tablets, [package insert]
Quesney-Huneeus, V., Galick, H. A., and Siperstein, M. D. (1983). The
dual role of mevalonate in the cell cycle. J. Biol. Chem. 258, 378-385.
Robison, R. L., Suter, W., and Cox, R. H. (1994). Carcinogenicity and
mutagenicity studies with fluvastatin, a new, entirely synthetic HMGCoA reductase inhibitor. Fundam. Appl. Toxicol. 23, 9-20.
Soma, M. R., Corsini, A , and Paoletti, R. (1992). Cholesterol and mevalonic
acid modulation in cell metabolism and multiplication. Toxicol. Lett. 64/
65, 1-15.
Surani, M. A. H., Kimber, S. J., and Osbom, J. C. (1983). Mevalonate reverses the developmental arrest of preimplantation mouse embryos by
Compactin, an inhibitor of HMGCoA reductase./ Embryol. Exp. Morph.
75, 205-223.
Tukey, J. W., Ciminera, J. L., and Heyse, J. F. (1985). Testing the statistical
certainty of a response to increasing doses of a drug. Biometrics 41, 2 9 5 301.
Wecker, J. R., Isor, J. R , and Foss, J. A. (1985). Reflex modification as a
test for sensory function Neurobehav. Toxicol. Teratol. 7, 733-738.
Wise, L. D., Majka, J. A., Robertson, R T , et al. (1990a). Simvastatin
(MK-0733): Oral teratogenicity study in rats pre- and postnatal observation. Oyo Yakuri 39, 143-158.
Wise, L. D., Minsker, D. H., Robertson, R. T., Bokelman, D. L., Akutsu,
S., and Fujii, T. (1990b). Simvastatin (MK-0733): Oral fertility study in
rats. Oyo Yakun 39, 127-141.
Yount, N. Y., and McNamara, D. J. (1991). Dietary regulation of maternal
and fetal cholesterol metabolism in the guinea pig. Bwchim. Bwphys.
Acta 1085, 82-90.