# Developmental and Reproductive Effects of SE5-OH: An Equol-Rich Soy-Based Ingredient

Authors: Ray A. Matulka; Ikuo Matsuura; Tohru Uesugi; Tomomi Ueno; George Burdock
Journal: Journal of Toxicology
 (2009)

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## Abstract

Consumption of the isoflavones daidzein, genistein, glycitein, and their structural analogues is generally considered beneficial to human health. Equol is not found in soy, but is converted from daidzein by human gut bacterial flora. Research indicates that between 30–50% of the population is capable of converting daidzein to equol; therefore, there has been recent development of a new equol-rich functional food that relies on bacterial conversion of daidzein to equol under strictly controlled conditions. Therefore, a new equol-rich soy product (SE5-OH) has been developed, based on the bacterial conversion of daidzein; and its reproductive and developmental toxicity has been evaluated in a two-generation study and a developmental toxicity study with Sprague-Dawley rats at dose levels of 200, 1000, and 2000 mg/kg/day by gavage. SE5-OH contains approximately 0.65% equol, 0.024% daidzein, 0.022% genistein, and 0.30% glycitein. From the reproductive study, the no-observed-adverse-effect-level (NOAEL) for SE5-OH determined for both male and female rats is 1000 mg/kg/day (6.5 mg equol/kg/day). In the developmental toxicity phase of the study, no effects by SE5-OH were found in the embryo-fetus at any of the doses tested. The NOAEL for developmental effects of SE5-OH is 2000 mg/kg/day (13 mg equol/kg/day).


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## Body

## 1. Introduction

Over the past two decades, scientific literature has indicated that consumption of soy products and, in particular, their isoflavone
components have been associated with the reduction of the risk of
cancer, coronary vascular disease, osteoporosis, and other pathologies that are
associated with menopause and ageing in both sexes [1–3]. One such
isoflavone is equol: a microbial metabolite of daidzein, which is structurally
related to 17β-estradiol [4]. Consumption of daidzein
in subjects that were found to be able to metabolize daidzein to equol has been
linked with delay in the onset of menopause and menopausal-associated symptoms,
markers of cardiovascular disease, and prostate cancer [5, 6]; however, there
is conflicting evidence concerning the ability to produce equol and subsequent
health benefits [6, 7]. Although there are known dietary sources of equol, the
levels of equol in these milk samples are relatively low (in the range of 60–450 ng equol/mL) [8, 9]. Therefore, the consumption of equol-enriched soy-based products may be of
health benefit to individuals who do not convert daidzein to equol.In nature, equol is formed exclusively by
the intestinal microbiota although chemical synthesis pathways have also been
described [10, 11]. The enzymatic process of producing equol from daidzein
resides with the gut microbial flora [12, 13], similar to the pathway presented
in Figure 1. Although it has been
estimated that there are more than 400 species of bacteria in the human gut
that may influence the normal structure and function of the intestinal system [14, 15], research indicates that, depending upon the population studied, between 30–50% of the
population is host to bacteria which metabolize daidzein into equol [6, 16, 17]. Recent research has indicated that humans that host bacteria, able to metabolize daidzein to equol, convert approximately 30–40% of daidzein into equol, and the types of food consumed may be one
of several factors that affect the ability to produce equol [18–20]. Serum levels of equol in
equol producers that consume daidzein have been noted at up to 76±137 nmol/L.Figure 1
Daidzein metabolism to equol.The content of equol in the soy-based
product (SE5-OH) has been enriched as a result of fermentation of soy germ with
the bacteriumL. garvieae (Otsuka
Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan). L.
garvieae is not a normal member of the human gut flora, but is typically
found in fish, and within fish aquacultures, and is associated with decreased
growth rates, an unmarketable appearance, and mortality, but is not known to
produce toxins [21]. L. garvieae is a
rare pathogen with a low virulence in humans with gastrointestinal disorders
that consumed raw fish, also has been found in several different cheeses, and may be an important fermentation microbe in the production of several cheeses, including artisanal Italian and traditional Egyptian Domiati cheeses [22–25]. The
controlled process of soy fermentation with L.
garvieae is capable of converting approximately 50% of the daidzein
contained within the soy to equol. SE5-OH is autoclaved to kill the bacteria, and
subsequently analyzed to ensure that SE5-OH does not contain any living L. garvieae; therefore, the live organism
will not be ingested by the consumer.Previously, we have reported acute and
subchronic toxicity studies of SE5-OH in the adult rat model, and determined
the NOAEL to be 2000 mg/kg/day following administration for 90 days [26]. The
objective of the studies described herein was to investigate the effect of the
specific equol-enriched soy-based SE5-OH product on rat development and
reproduction over two generations.


## 2. Methods and Materials

### 2.1. Test Articles

The test article, referred to as SE5-OH, was
prepared by a controlled fermentation of a daidzein-rich soy germ containing
isoflavones withL. garvieae (Otsuka
Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan). SE5-OH contains approximately 0.65% equol, 0.024%
daidzein, 0.022% genistein, and 0.30% glycitein. The test article is a light
brown powder that includes five specific isoflavones; the amounts in the two
lots of test article used in these studies are presented in Table 1. The
test article control was hydroxypropylmethylcellulose (HPMC), supplied by Shin-Etsu
Chemical Co., Ltd (Chiyoda-ku, Tokyo, Japan).Table 1
Isoflavone constituents of SE5-OH.
Isoflavoneingredient*Reproductive toxicity study(Lot060106P)** (mg/g)Reproductive toxicity study and developmental toxicity study(Lot051206P)**(mg/g)*Daidzein0.323±0.00150.275±0.0025Genistein0.293±0.00170.287±0.0032Glycitein1.65±0.0152.04±0.010Dihydrodaidzein0.362±0.00210.337±0.0036Equol5.16±0.0705.24±0.025The* amount of each isoflavone ingredient is the average of three separate measurements, mean ± standard deviation.
Both** lots were used in the reproductive study, while only Lot 051206P was used in the developmental study.


### 2.2. Animals

Male and/or female Crl:CD(SD) rats, supplied
by the Atsugi Breeding Center of Charles River Laboratories (CRL) Japan, Inc., (Kohoku-ku,
Yokohama, Japan) were used for both the
developmental and reproductive toxicity studies. All animals were clinically
monitored at the time of delivery to the testing facility and during the
acclimation period. Housing consisted of stainless steel or polycarbonate cages, with a twelve-hour light period (7:00  AM to
7:00  PM). Temperature was maintained at20.1–23.4°C with a relative humidity of 49.6–69.2% and 6–20 air changes/h. The bedding, which was used
only in the reproductive toxicity study, was of the Beta-Chip brand supplied by Charles River Laboratories Japan, Inc. The rats were six weeks of age at the start of dosing in the reproductive
toxicity study and twelve weeks of age at mating in the developmental toxicity
study. Rats were acclimatized for five or six days before use.


### 2.3. Guidelines

The developmental toxicity and reproductive toxicity studies were carried out under the guidelines ofRedbook 2000 and FDA’s Toxicological Principles for the Safety Assessment of Food Ingredients [27, 28]. Both studies were conducted within the Good Laboratory Practice (GLP) standards for the conduction of nonclinical studies on the safety of drugs (Ministry of Health and Welfare (MHW), Japan, Ordinance Number 21, 26 March 1997, [http://pdg-a258.koyosha.co.jp/jsqa2/en/glp/glp424e.html]). This study was approved by the Committee of Kashima Laboratory for Ethics in Animal Studies in compliance with “Guidelines for Animals Studies (Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Kamisu-shi, Ibaraki, Japan).”


### 2.4. Experimental Design

#### 2.4.1. Developmental Toxicity Study

(1) Exposure and Observations
On
gestational days (GDs) 6–19, three groups
of 22 pregnant females were administered doses of SE5-OH in 1% aqueous HPMC by
gavage at levels of 200, 1000, and 2000 mg/kg/day, respectively; the control
group (22 pregnant females) was administered 1% HPMC vehicle only. Animals were
observed twice daily for clinical signs during the dosing period, and once
daily following the dosing regimen. Body weights and gross weights of the diets
were measured on GD 0, 6, 8, 10, 12, 14, 16, 18, and 20. Body weight gain was
calculated on the basis of body weights measured on GD 6.(2) Necropsy
On GD 20, dams were euthanized, the gravid
uterus was collected from each dam and weighed to allow for calculation of
corrected body weight and corrected body weight gain. Corrected body weight was
determined by subtracting the gravid uterine weight from the body weight on GD
20, while the corrected body weight gain was calculated by subtracting the body
weight on GD 6 from the corrected body weight. At necropsy of the dams,
thoracic and abdominal organs and tissues were examined macroscopically. Any
organs or tissues showing abnormalities were collected and preserved. If such
collections were conducted,
the same tissues were collected from three control dams and also preserved.
Ovaries and uterus were removed and examined for the number of corpora lutea,
implantations, live fetuses, and dead embryos. Early embryonic deaths were
considered to be represented by the number of implantation sites plus placental
remnants; late embryonic deaths were represented by macerated embryos plus dead
fetuses.
Live fetuses were weighed, euthanized, sexed,
and examined (external and oral cavity examinations). Approximately half of the
live fetuses in each litter from the control and the 2000 mg/kg dose dams were
subjected to visceral examination, which included the head, neck, thorax, and
abdomen. The remaining fetuses were examined for skeletal anomalies and degrees
of ossification in cervical vertebral bodies, sternebrae, metacarpi, metatarsi,
and sacral/caudal vertebral bodies.


#### 2.4.2. Reproductive Toxicity Study

Groups of 52 rats (26/sex/group) served as
theF0 generation and were administered the same SE5-OH test article at the same doses via
gavage as in the developmental
toxicity study (i.e., 0, 200,
1000, and 2000 mg/kg/day). Dosing commenced at six weeks of age and lasted for
ten weeks before mating and then throughout the mating period. The mating
procedure involved the placement of one male and one female in the same cage
for a maximum of two weeks, with
reproductive capacity judged by copulation index, fertility index, precoital
period, gestation period, and gestation index. F0 females that became
pregnant continued on this exposure regimen through gestation and lactation
until weaning of the F1 animals. The F1 generation
males and females were also administered the same dosing regimen of SE5-OH,
beginning on postnatal day (PND)
21, for ten to eleven weeks before
mating and throughout the mating period, with pregnant females continuing on
the dosing schedules through gestation and lactation until weaning of the F2 generation. Throughout this entire process, animals were observed twice daily
for clinical signs; body weights and food consumption were measured weekly
until the start of gestation in both F0 and F1 generations. Estrous cycle was examined for three weeks before
the start of mating in both F0 and F1 females.
During gestation, females in both generations were weighed on GD 0, 3, 7, 10, 14, 17, and 20 and then on lactation
day (LD) 0, 4, 7, 10, 14, 17, and 21 during
lactation. Food consumption was also measured during GD 0, 7, 14, and 20 and on
LD 0, 4, 7, 14, and 21. On the day after the animals reached the end of their respective
exposure periods, they were euthanized for gross necropsy. The following
tissues were weighed and preserved: adrenals, brain, kidney, liver, female
reproductive tissues (uterus, ovaries), male reproductive tissues (testes,
epididymis, prostate, and seminal
vesicles including coagulating glands), pituitary, spleen, thymus (neonatal only),
and thyroid gland. Histological examinations were conducted on tissues from control
and high- (2000 mg/kg) dose groups only and included adrenals (F0 females were examined in all dose groups), male and female reproductive organs,
pituitary, and liver tissues from F0 females. Sperm was also collected from the cauda epididymis of all F0 and F1 males and evaluated for motility, and the sperm from the control and 2000 mg/kg dose group
were also evaluated for spermatid cells (testis), sperm number, and morphology.Clinical parameters for offspring included
examination for external anomalies, sex ratios, live birth index (i.e., number of live pups/number of
total pups born), as well as viability index on PND 4 and weaning index on PND
21; pup weights were measured at birth as well as PND 4, 7, 14, and 21. On PND
4, litter sizes were adjusted randomly to eight pups per dam. Pups were
also examined for physical and functional developmental parameters, which
included incisor eruption, eye opening, reflexes for righting, auditory startle,
pain, corneal, and pupillary reactions. Sexual maturation was determined inF1 rats by recording the age and weight on the day of vaginal opening after PND 27 was reached or when
balanopreputial separation from PND
35 was recorded. At gross necropsy of F1 and F2 weanlings, brain, spleen, and thymus were collected from selected animals for
preservation (one male and one female selected from each litter in numerical
order of the offspring number in each litter, which was randomly assigned).
Ovaries, vagina, and uterus were collected from females, while testes,
epididymis, seminal vesicles, and prostate were collected from males. Organ
weights were determined from one animal of each sex per litter for brain, thymus, spleen, and
uterus.


### 2.5. Statistical Analyses

Statistical analysis for the reproductive and developmental toxicity
study was conducted using several different tests, using the toxicological data
processing system (MiTOX, Mitsui Zosen Systems Research, Inc., Japan). The
data of offspring obtained before weaning were analyzed on the basis of litter
mean values except for the sex ratio. The normality of the data was assumed,
unless otherwise indicated. Homogeneity of variance was tested by Bartlett’s test; when the data
were homogeneous, a one-way analysis of variance (ANOVA) was performed for
statistical comparison. Heterogeneous data were tested by the Kruskal-Wallis test. When a significant intergroup
difference was found, Dunnett's or the Dunnett-type multiple comparison test
was used. For some of the data (e.g., days until copulation, number of estrus
stages without copulation, gestation length, sperm motility, birth index,
viability index on PND 0, 4, and 21, incidence of offspring with
external anomalies, postnatal physical development, reflex and sensory
function, as well as sexual maturation (vaginal opening and cleavage of
balanopreputial gland)), the Kruskal-Wallis test was applied first, and when a
significant intergroup difference was found, the Dunnett-type multiple
comparison test was used. The multiple comparison test was utilized to evaluate
statistical significance of changes in body weight, body weight gain, food
consumption, organ weights, number of implantations, number of offspring,
number of live offspring, and estrous cycle lengths. However, for the spermatid
count and sperm count, the homogeneity of variance was tested by the F test, and
when the variance was homogeneous, Student’st-test was performed for
the statistical comparison. When heterogeneous, the Aspin-Welch t-test
was used. Moreover, Wilcoxon’s rank sum test was used for comparison of the
incidence of abnormal sperm (morphologically abnormal sperm and tailless sperm), visceral examination,
and skeletal
examinations. The categorical data (e.g., copulation index, fertility index,
gestation index, sex ratio (male/female), incidence of females with irregular
estrous cycles, and dams with external abnormal offspring) were analyzed by
Fisher’s exact probability test. All statistical tests used two-tailed significance
level of 5%.


## 2.1. Test Articles

The test article, referred to as SE5-OH, was
prepared by a controlled fermentation of a daidzein-rich soy germ containing
isoflavones withL. garvieae (Otsuka
Pharmaceutical Co., Ltd., Chiyoda-ku, Tokyo, Japan). SE5-OH contains approximately 0.65% equol, 0.024%
daidzein, 0.022% genistein, and 0.30% glycitein. The test article is a light
brown powder that includes five specific isoflavones; the amounts in the two
lots of test article used in these studies are presented in Table 1. The
test article control was hydroxypropylmethylcellulose (HPMC), supplied by Shin-Etsu
Chemical Co., Ltd (Chiyoda-ku, Tokyo, Japan).Table 1
Isoflavone constituents of SE5-OH.
Isoflavoneingredient*Reproductive toxicity study(Lot060106P)** (mg/g)Reproductive toxicity study and developmental toxicity study(Lot051206P)**(mg/g)*Daidzein0.323±0.00150.275±0.0025Genistein0.293±0.00170.287±0.0032Glycitein1.65±0.0152.04±0.010Dihydrodaidzein0.362±0.00210.337±0.0036Equol5.16±0.0705.24±0.025The* amount of each isoflavone ingredient is the average of three separate measurements, mean ± standard deviation.
Both** lots were used in the reproductive study, while only Lot 051206P was used in the developmental study.


## 2.2. Animals

Male and/or female Crl:CD(SD) rats, supplied
by the Atsugi Breeding Center of Charles River Laboratories (CRL) Japan, Inc., (Kohoku-ku,
Yokohama, Japan) were used for both the
developmental and reproductive toxicity studies. All animals were clinically
monitored at the time of delivery to the testing facility and during the
acclimation period. Housing consisted of stainless steel or polycarbonate cages, with a twelve-hour light period (7:00  AM to
7:00  PM). Temperature was maintained at20.1–23.4°C with a relative humidity of 49.6–69.2% and 6–20 air changes/h. The bedding, which was used
only in the reproductive toxicity study, was of the Beta-Chip brand supplied by Charles River Laboratories Japan, Inc. The rats were six weeks of age at the start of dosing in the reproductive
toxicity study and twelve weeks of age at mating in the developmental toxicity
study. Rats were acclimatized for five or six days before use.


## 2.3. Guidelines

The developmental toxicity and reproductive toxicity studies were carried out under the guidelines ofRedbook 2000 and FDA’s Toxicological Principles for the Safety Assessment of Food Ingredients [27, 28]. Both studies were conducted within the Good Laboratory Practice (GLP) standards for the conduction of nonclinical studies on the safety of drugs (Ministry of Health and Welfare (MHW), Japan, Ordinance Number 21, 26 March 1997, [http://pdg-a258.koyosha.co.jp/jsqa2/en/glp/glp424e.html]). This study was approved by the Committee of Kashima Laboratory for Ethics in Animal Studies in compliance with “Guidelines for Animals Studies (Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Kamisu-shi, Ibaraki, Japan).”


## 2.4. Experimental Design

### 2.4.1. Developmental Toxicity Study

(1) Exposure and Observations
On
gestational days (GDs) 6–19, three groups
of 22 pregnant females were administered doses of SE5-OH in 1% aqueous HPMC by
gavage at levels of 200, 1000, and 2000 mg/kg/day, respectively; the control
group (22 pregnant females) was administered 1% HPMC vehicle only. Animals were
observed twice daily for clinical signs during the dosing period, and once
daily following the dosing regimen. Body weights and gross weights of the diets
were measured on GD 0, 6, 8, 10, 12, 14, 16, 18, and 20. Body weight gain was
calculated on the basis of body weights measured on GD 6.(2) Necropsy
On GD 20, dams were euthanized, the gravid
uterus was collected from each dam and weighed to allow for calculation of
corrected body weight and corrected body weight gain. Corrected body weight was
determined by subtracting the gravid uterine weight from the body weight on GD
20, while the corrected body weight gain was calculated by subtracting the body
weight on GD 6 from the corrected body weight. At necropsy of the dams,
thoracic and abdominal organs and tissues were examined macroscopically. Any
organs or tissues showing abnormalities were collected and preserved. If such
collections were conducted,
the same tissues were collected from three control dams and also preserved.
Ovaries and uterus were removed and examined for the number of corpora lutea,
implantations, live fetuses, and dead embryos. Early embryonic deaths were
considered to be represented by the number of implantation sites plus placental
remnants; late embryonic deaths were represented by macerated embryos plus dead
fetuses.
Live fetuses were weighed, euthanized, sexed,
and examined (external and oral cavity examinations). Approximately half of the
live fetuses in each litter from the control and the 2000 mg/kg dose dams were
subjected to visceral examination, which included the head, neck, thorax, and
abdomen. The remaining fetuses were examined for skeletal anomalies and degrees
of ossification in cervical vertebral bodies, sternebrae, metacarpi, metatarsi,
and sacral/caudal vertebral bodies.


### 2.4.2. Reproductive Toxicity Study

Groups of 52 rats (26/sex/group) served as
theF0 generation and were administered the same SE5-OH test article at the same doses via
gavage as in the developmental
toxicity study (i.e., 0, 200,
1000, and 2000 mg/kg/day). Dosing commenced at six weeks of age and lasted for
ten weeks before mating and then throughout the mating period. The mating
procedure involved the placement of one male and one female in the same cage
for a maximum of two weeks, with
reproductive capacity judged by copulation index, fertility index, precoital
period, gestation period, and gestation index. F0 females that became
pregnant continued on this exposure regimen through gestation and lactation
until weaning of the F1 animals. The F1 generation
males and females were also administered the same dosing regimen of SE5-OH,
beginning on postnatal day (PND)
21, for ten to eleven weeks before
mating and throughout the mating period, with pregnant females continuing on
the dosing schedules through gestation and lactation until weaning of the F2 generation. Throughout this entire process, animals were observed twice daily
for clinical signs; body weights and food consumption were measured weekly
until the start of gestation in both F0 and F1 generations. Estrous cycle was examined for three weeks before
the start of mating in both F0 and F1 females.
During gestation, females in both generations were weighed on GD 0, 3, 7, 10, 14, 17, and 20 and then on lactation
day (LD) 0, 4, 7, 10, 14, 17, and 21 during
lactation. Food consumption was also measured during GD 0, 7, 14, and 20 and on
LD 0, 4, 7, 14, and 21. On the day after the animals reached the end of their respective
exposure periods, they were euthanized for gross necropsy. The following
tissues were weighed and preserved: adrenals, brain, kidney, liver, female
reproductive tissues (uterus, ovaries), male reproductive tissues (testes,
epididymis, prostate, and seminal
vesicles including coagulating glands), pituitary, spleen, thymus (neonatal only),
and thyroid gland. Histological examinations were conducted on tissues from control
and high- (2000 mg/kg) dose groups only and included adrenals (F0 females were examined in all dose groups), male and female reproductive organs,
pituitary, and liver tissues from F0 females. Sperm was also collected from the cauda epididymis of all F0 and F1 males and evaluated for motility, and the sperm from the control and 2000 mg/kg dose group
were also evaluated for spermatid cells (testis), sperm number, and morphology.Clinical parameters for offspring included
examination for external anomalies, sex ratios, live birth index (i.e., number of live pups/number of
total pups born), as well as viability index on PND 4 and weaning index on PND
21; pup weights were measured at birth as well as PND 4, 7, 14, and 21. On PND
4, litter sizes were adjusted randomly to eight pups per dam. Pups were
also examined for physical and functional developmental parameters, which
included incisor eruption, eye opening, reflexes for righting, auditory startle,
pain, corneal, and pupillary reactions. Sexual maturation was determined inF1 rats by recording the age and weight on the day of vaginal opening after PND 27 was reached or when
balanopreputial separation from PND
35 was recorded. At gross necropsy of F1 and F2 weanlings, brain, spleen, and thymus were collected from selected animals for
preservation (one male and one female selected from each litter in numerical
order of the offspring number in each litter, which was randomly assigned).
Ovaries, vagina, and uterus were collected from females, while testes,
epididymis, seminal vesicles, and prostate were collected from males. Organ
weights were determined from one animal of each sex per litter for brain, thymus, spleen, and
uterus.


## 2.4.1. Developmental Toxicity Study

(1) Exposure and Observations
On
gestational days (GDs) 6–19, three groups
of 22 pregnant females were administered doses of SE5-OH in 1% aqueous HPMC by
gavage at levels of 200, 1000, and 2000 mg/kg/day, respectively; the control
group (22 pregnant females) was administered 1% HPMC vehicle only. Animals were
observed twice daily for clinical signs during the dosing period, and once
daily following the dosing regimen. Body weights and gross weights of the diets
were measured on GD 0, 6, 8, 10, 12, 14, 16, 18, and 20. Body weight gain was
calculated on the basis of body weights measured on GD 6.(2) Necropsy
On GD 20, dams were euthanized, the gravid
uterus was collected from each dam and weighed to allow for calculation of
corrected body weight and corrected body weight gain. Corrected body weight was
determined by subtracting the gravid uterine weight from the body weight on GD
20, while the corrected body weight gain was calculated by subtracting the body
weight on GD 6 from the corrected body weight. At necropsy of the dams,
thoracic and abdominal organs and tissues were examined macroscopically. Any
organs or tissues showing abnormalities were collected and preserved. If such
collections were conducted,
the same tissues were collected from three control dams and also preserved.
Ovaries and uterus were removed and examined for the number of corpora lutea,
implantations, live fetuses, and dead embryos. Early embryonic deaths were
considered to be represented by the number of implantation sites plus placental
remnants; late embryonic deaths were represented by macerated embryos plus dead
fetuses.
Live fetuses were weighed, euthanized, sexed,
and examined (external and oral cavity examinations). Approximately half of the
live fetuses in each litter from the control and the 2000 mg/kg dose dams were
subjected to visceral examination, which included the head, neck, thorax, and
abdomen. The remaining fetuses were examined for skeletal anomalies and degrees
of ossification in cervical vertebral bodies, sternebrae, metacarpi, metatarsi,
and sacral/caudal vertebral bodies.


## 2.4.2. Reproductive Toxicity Study

Groups of 52 rats (26/sex/group) served as
theF0 generation and were administered the same SE5-OH test article at the same doses via
gavage as in the developmental
toxicity study (i.e., 0, 200,
1000, and 2000 mg/kg/day). Dosing commenced at six weeks of age and lasted for
ten weeks before mating and then throughout the mating period. The mating
procedure involved the placement of one male and one female in the same cage
for a maximum of two weeks, with
reproductive capacity judged by copulation index, fertility index, precoital
period, gestation period, and gestation index. F0 females that became
pregnant continued on this exposure regimen through gestation and lactation
until weaning of the F1 animals. The F1 generation
males and females were also administered the same dosing regimen of SE5-OH,
beginning on postnatal day (PND)
21, for ten to eleven weeks before
mating and throughout the mating period, with pregnant females continuing on
the dosing schedules through gestation and lactation until weaning of the F2 generation. Throughout this entire process, animals were observed twice daily
for clinical signs; body weights and food consumption were measured weekly
until the start of gestation in both F0 and F1 generations. Estrous cycle was examined for three weeks before
the start of mating in both F0 and F1 females.
During gestation, females in both generations were weighed on GD 0, 3, 7, 10, 14, 17, and 20 and then on lactation
day (LD) 0, 4, 7, 10, 14, 17, and 21 during
lactation. Food consumption was also measured during GD 0, 7, 14, and 20 and on
LD 0, 4, 7, 14, and 21. On the day after the animals reached the end of their respective
exposure periods, they were euthanized for gross necropsy. The following
tissues were weighed and preserved: adrenals, brain, kidney, liver, female
reproductive tissues (uterus, ovaries), male reproductive tissues (testes,
epididymis, prostate, and seminal
vesicles including coagulating glands), pituitary, spleen, thymus (neonatal only),
and thyroid gland. Histological examinations were conducted on tissues from control
and high- (2000 mg/kg) dose groups only and included adrenals (F0 females were examined in all dose groups), male and female reproductive organs,
pituitary, and liver tissues from F0 females. Sperm was also collected from the cauda epididymis of all F0 and F1 males and evaluated for motility, and the sperm from the control and 2000 mg/kg dose group
were also evaluated for spermatid cells (testis), sperm number, and morphology.Clinical parameters for offspring included
examination for external anomalies, sex ratios, live birth index (i.e., number of live pups/number of
total pups born), as well as viability index on PND 4 and weaning index on PND
21; pup weights were measured at birth as well as PND 4, 7, 14, and 21. On PND
4, litter sizes were adjusted randomly to eight pups per dam. Pups were
also examined for physical and functional developmental parameters, which
included incisor eruption, eye opening, reflexes for righting, auditory startle,
pain, corneal, and pupillary reactions. Sexual maturation was determined inF1 rats by recording the age and weight on the day of vaginal opening after PND 27 was reached or when
balanopreputial separation from PND
35 was recorded. At gross necropsy of F1 and F2 weanlings, brain, spleen, and thymus were collected from selected animals for
preservation (one male and one female selected from each litter in numerical
order of the offspring number in each litter, which was randomly assigned).
Ovaries, vagina, and uterus were collected from females, while testes,
epididymis, seminal vesicles, and prostate were collected from males. Organ
weights were determined from one animal of each sex per litter for brain, thymus, spleen, and
uterus.


## 2.5. Statistical Analyses

Statistical analysis for the reproductive and developmental toxicity
study was conducted using several different tests, using the toxicological data
processing system (MiTOX, Mitsui Zosen Systems Research, Inc., Japan). The
data of offspring obtained before weaning were analyzed on the basis of litter
mean values except for the sex ratio. The normality of the data was assumed,
unless otherwise indicated. Homogeneity of variance was tested by Bartlett’s test; when the data
were homogeneous, a one-way analysis of variance (ANOVA) was performed for
statistical comparison. Heterogeneous data were tested by the Kruskal-Wallis test. When a significant intergroup
difference was found, Dunnett's or the Dunnett-type multiple comparison test
was used. For some of the data (e.g., days until copulation, number of estrus
stages without copulation, gestation length, sperm motility, birth index,
viability index on PND 0, 4, and 21, incidence of offspring with
external anomalies, postnatal physical development, reflex and sensory
function, as well as sexual maturation (vaginal opening and cleavage of
balanopreputial gland)), the Kruskal-Wallis test was applied first, and when a
significant intergroup difference was found, the Dunnett-type multiple
comparison test was used. The multiple comparison test was utilized to evaluate
statistical significance of changes in body weight, body weight gain, food
consumption, organ weights, number of implantations, number of offspring,
number of live offspring, and estrous cycle lengths. However, for the spermatid
count and sperm count, the homogeneity of variance was tested by the F test, and
when the variance was homogeneous, Student’st-test was performed for
the statistical comparison. When heterogeneous, the Aspin-Welch t-test
was used. Moreover, Wilcoxon’s rank sum test was used for comparison of the
incidence of abnormal sperm (morphologically abnormal sperm and tailless sperm), visceral examination,
and skeletal
examinations. The categorical data (e.g., copulation index, fertility index,
gestation index, sex ratio (male/female), incidence of females with irregular
estrous cycles, and dams with external abnormal offspring) were analyzed by
Fisher’s exact probability test. All statistical tests used two-tailed significance
level of 5%.


## 3. Results

### 3.1. Developmental Toxicity Study

#### 3.1.1. Effects on Dams

With the exception of a transient lack of
weight gain (see Figure2), no abnormal
clinical signs were observed in any of the dams. This included observations for
death, moribundity, abortion, or premature delivery (data not shown). In dams
exposed to 2000 mg/kg, significant decreases in body weight gain were noted on
GD 8–16 but these
values had returned to control levels by GD 20. The same transient lack of
weight gain was noted for dams in the 1000 mg/kg exposure group, but only on GD
8. No treatment-related effects were seen for food consumption, gravid uterine
weights, corrected body weight, and corrected body weight gain; and there were
no abnormalities seen at necropsy (data not shown).Figure 2
Changes in body weight
gain (grams) observed in dams exposed to SE5-OH during gestational days 0–20 during the
developmental toxicity study in rats; significant when compared to control:P*<.05; P**<.01


#### 3.1.2. Effects on Embryo-Fetal Development

In all treated groups, there were no
significant differences in number of corpora lutea, implantations, dead
embryos, postimplantation losses, live fetuses, fetal deaths, fetal weight, or sex ratio. Several
isolated embryo-fetal effects were noted during the placental observations, and
external, visceral, and skeletal examinations. These changes are summarized in Table2.Table 2
Isolated embryo-fetal effects noted during
necropsy observations/examinations in the developmental toxicity study in rats.
EffectOccurrenceFusion
of placentaeOccurred in one dam in the 200 mg/kg dose group. No other
placental abnormalities identified in any group.Rudimentary
tailOccurred in one fetus of the 1000 mg/kg dose group. No
other external fetal abnormalities identified in any group.Visceral
anomaly: ventricular septal
defectOccurred in one control group fetus and one 2000 mg/kg
group fetus. No other anomalies identified in any group.Visceral variants: thymic neck remnant, supernumerary
coronary ostium, left umbilical artery, and renal pelvis dilationOccurred
with equal incidence in the control and 2000 mg/kg groups.Skeletal
anomalies/malformations: wavy and nodulated ribsOccurred in one 2000 mg/kg group fetus. No other anomalies
identified in any group.Skeletal variants: foramen hypoglossi double, closure of
transverse foramen of one or more cervical vertebral arches, transverse foramen opening in 7th
cervical arch, splitting of ossification centers in thoracic vertebral
bodies, hemicentric thoracic centrum, dumbbell ossification of thoracic
centrum, lumbarization, cervical ribs, short supernumerary 14th rib, reduced
13th ribs, splitting of sternebraeIncidence
of dumbbell ossification of thoracic centrum was significantly low in the
2000 mg/kg dosing group but was judged incidental with no toxicological
significance.


### 3.2. Reproductive Toxicity Study

#### 3.2.1. Parental Animals

Transient
decrease of weight gain and reduced food intake in theF0 and F1 females of 2000 mg/kg/day, and histological change of adrenal cortex (a
diffuse increase in lipid droplets in the zona glomerulosa) in the F0 females of 2000 mg/kg/day were noted (see Table 3). Food intake was reduced in the F0 females in the 1000 mg/kg/day group. In addition, increased liver weight in the F0 female of 1000 and
2000 mg/kg/day dose groups, increased adrenal weight in the F0 males
at the 2000 mg/kg/day dose group, as well as increased thyroid weight in the F1 males (see Table 4) was noted. No adverse clinical signs
(including death, moribundity, abortion, or premature delivery), necropsy findings, or organ weights
were observed in any of the F0 and F1 parental animals.Table 3
Body weight and food consumption in male and female Sprague-Dawley rats during the
two-generation reproductive toxicity study.
GenerationF0F1Dose (mg/kg/day)GenderPeriod (days)020010002000020010002000Body weight (g)MalePremating0195.9±6.6196.5±5.7196.2±6.3195.5±6.259.6±4.9#59.7±5.059.4±5.757.1±5.170540.0±47.2560.1±56.6534.3±46.4539.3±41.2451.8±35.1461.±32.1442.8±37.8445.5±34.9(n=26)(n=26)(n=26)(n=26)(n=23)(n=23)(n=21)(n=22)FemalePremating0152.2±8.5152.5±6.9151.0±6.9151.2±6.855.4±6.057.0±5.857.6±4.854.8±5.570303.9±35.6298.3±21.5289.2±22.4283.0±19.1*266.6±22.9270.0±26.8263.2±21.0252.9±17.1(n=26)(n=26)(n=26)(n=26)(n=24)(n=23)(n=20)(n=22)Gestation7337.9±28.8333.9±21.3323.3±22.7318.5±21*358.9±32.3367.1±34.9352.6±29.6334.7±20.7*14371.0±29.5364.0±23.3355.4±23.8349.3±23.4*395.4±34.4400.9±35.5383.4±29.6367.6±22.9*20443.0±36.4440.2±25.4434.7±26.7428.9±23.6474.9±40.5473.3±39.1453.9±38.9437.3±34.9**(n=25)(n=24)(n=22)(n=22)(n=18)(n=19)(n=19)(n=21)Lactation21355.0±24.2345.8±20.4345.3±21.7343.0±19.5365.3±25.6370.8±28.0368.0±22.8355.8±14.8(n=24)(n=23)(n=21)(n=22)(n=18)(n=17)(n=18)(n=19)Body weight gain (g)MalePremating0–70344.1±45.1363.6±54.4338.1±43.7343.8±40.0392.3±34.1401.8±30.2383.4±34.1388.4±32.1FemalePremating0–70151.7±31.1145.9±20.4138.2±19.3131.9±16.5*211.2±20.8213.0±25.0205.4±19.3198.1±16.9Gestation0–733.2±7.131.3±5.529.6±8.930.7±7.936.8±7.734.6±8.732.1±9.333.3±7.90–1466.3±12.761.4±8.861.7±8.061.5±12.473.2±8.468.4±12.162.8±10.7*66.2±11.80–20138.3±22.3137.6±16.4141.0±11.0141.0±18.5152.8±13.0140.8±24.5133.3±20.8**136.0±26.5Lactation0–211.6±20.3−2.4±20.26.8±20.83.4±13.9−20.3±20.5−17.4±25.4−7.7±23.4−5.5±23.0Food consumption (g)MalePremating7026.2±1.926.9±1.825.4±1.625.1±1.830.5±2.031.3±1.829.0±1.828.9±1.9FemalePremating7019.0±1.718.5±1.617.6±1.2*16.3±0.9**20.3±2.120.6±1.519.2±1.317.5±1.1**Gestation722.6±1.722.1±1.920.7±2.1**20.4±2.4**24.4±1.924.3±2.922.6±3.121.4±2.1**1424.8±2.424.2±2.223.1±2.0*22.8±2.3**26.3±2.226.3±2.824.7±2.623.7±2.6**2026.0±2.425.7±2.626.0±2.225.1±2.127.7±2.126.3±2.525.8±2.126.4±2.6Lactation2168.4±8.568.8±3.769.4±4.067.4±5.467.2±6.667.9±5.364.1±6.465.7±9.2Mean ± standard
deviation; significantly different from control:P<.05*, P<.01**; # the start of dosing for the
F0
groups
was at six weeks of age. The start of dosing for the
F1
group was on day 21
postparturition, to maintain SE5-OH ingestion via lactation; n: number of male and female rats per dose
group.Table 4
Organ weight changes in male and female breeding Sprague-Dawley rats in theF0 and F1 generations of the two-generation rat study.
GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=22)200 (n=22)1000 (n=20)2000 (n=26)MaleThyroids,
absolute organ weight(mg)A30.01±5.45C31.65±5.9429.94±3.8830.32±3.9530.19±4.4831.89±4.9135.15±5.27**32.09±4.18Thyroids,
relative organ weight(mg)B5.23±0.685.33±0.865.33±0.735.34±0.784.55±0.614.76±0.735.48±0.90**4.94±0.58Adrenals, absolute organ weight (mg)61.81±7.0563.95±10.1659.48±7.4068.90±9.01**62.18±7.3063.96±5.8357.61±7.5262.28±9.25Adrenals, relative
organ weight (mg)10.88±1.3110.82±1.6810.57±1.2812.10±1.58**9.41±1.149.56±1.078.95±1.089.62±1.57Seminal Vesicles, absolute organ weight
(g)3.11±0.332.90±0.373.12±0.373.05±0.343.40±0.293.26±0.443.23±0.393.13±0.37Seminal
vesicles, relative organ weight (g)0.55±0.070.49±0.09*0.55±0.080.54±0.060.52±0.080.49±0.010.51±0.080.48±0.06GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=18)200 (n=17)1000 (n=18)2000 (n=19)FemaleLiver,
absolute organ weight (g)13.70±1.3013.99±1.0814.39±1.6214.48±1.1115.01±1.2815.45±1.8616.02±1.4615.71±1.35Liver,
relative organ weight (g)3.86±0.324.05±0.314.17±0.35**4.23±0.29**4.12±0.344.17±0.414.36±0.334.42±0.33*Significantly different from control:P<.05*,
P<.01**; absoluteA organ weight is the weight of the
organ; relativeB organ weight is the weight of the organ relative
to the body weight at necropsy (body weight ratio); meanC ± standard deviation; n:
number of male and female rats per dose group.There were no adverse reproductive effects observed in any treatment group ofF0 and F1 animals
in terms of estrous cycle, copulation
index, fertility index, or precoital period (see Tables 5 and 6). No
adverse effects were seen in gestation period, gestation index, or parturition. No effect on implantation
number was found in the F0 generation although the number of
implantations was slightly, but
significantly, reduced in the F1 dams at 2000 mg/kg/day, there were no effects on implantation in the 200 and 1000 mg/kg/da F1 generation (see Tables 7
and 8). The decrease in
implantation number in the high dose did not significantly affect the birth
index or the number of offspring born alive. SE5-OH administration to male rats
did not affect any of the sperm parameters evaluated, which included sperm
motility, percentage and type of abnormal sperm, tailless sperm, sperm count,
or the number of homogenization-resistant sperm (see Table 9).Table 5
Effect
of SE5-OH administration on the reproductive performance of theF0 generation
rat dams.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.26±0.43#0.0±0.02.5±1.8100.096.2(n=25)1/26(n=26)(n=26)(26/26)(25/26)2004.18±0.320.0±0.02.8±1.692.3100.0(n=24)2/26(n=26)(n=24)(24/26)(24/24)10004.21±0.400.0±0.02.9±2.792.391.7(n=26)0/26(n=26)(n=24)(24/26)(22/24)20004.15±0.380.0±0.03.3±2.596.288.0(n=24)2/26(n=26)(n=25)(25/26)(22/25)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 6
Effect of SE5-OH
administration on theF1 generation rat dam reproductive performance.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.36±0.46#0.1±0.63.1±2.895.585.7(n=24)0/24(n=22)(n=21)(21/22)(18/21)2004.31±0.450.2±0.72.8±1.895.786.4(n=21)2/23(n=23)(n=22)(22/23)(19/22)10004.39±0.410.1±0.22.3±1.3100.095.0(n=20)0/20(n=20)(n=20)(20/20)(19/20)20004.30±0.430.0±0.02.5±1.5100.095.5(n=21)1/22(n=22)(n=22)(22/22)(21/22)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 7
Effects of SE5-OH administration onF0 generation rat dam gestation
and delivery parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.3±0.515.0±3.913.8±4.187.12±21.6496n2425252520022.2±0.415.1±3.613.8±3.489.03±20.4895.8n23242424100022.3±0.615.3±1.713.6±3.389.27±19.68100.0n22222222200022.0±0.314.7±2.113.8±2.393.63±9.28100.0n22222222Significantly different from control:P<.05*,
P<.01**; birthA index = (number of offspring born
alive divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 8
Effects of SE5-OH
administration onF1 generation rat dam gestation and delivery
parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.2±0.415.8±1.214.9±1.993.8±7.4100n=18n=18n=18n=1820022.2±0.415.8±2.514.0±2.388.9±9.2100n=18n=18n=18n=18100022.2±0.413.8±4.112.7±3.988.1±22.894.7n=18n=19n=19n=19200022.3±0.612.6±4.7*11.3±5.184.5±24.895.2n=20n=21n=21n=21BirthA index = (number of offspring born alive
divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 9
Sperm parameters
in control and high-dose male rats used as breeding males in theF0 and
F1 two-generation reproduction study.
Dose (mg/kg/day)Sperm motility (%)HRS (×106/g)Sperm count (cauda epididymal) (×106/g)Abnormal sperm (%)Types of abnormal sperm (%) no.Tailless sperm (%)ABCDEF0 generation091.0±4.583.73±12.52534.73±72.790.81±1.260.25±0.620.04±0.140.00±0.000.42±0.760.10±0.401.96±2.03n26262626262626262626200091.3±3.283.64±10.21537.29±66.920.33±0.370.04±0.140.00±0.000.00±0.000.29±0.350.00±0.001.81±2.83n26262626262626262626F1 generation086.0±20.175.98±17.86551.21±151.940.52±0.830.31±0.430.00±0.000.00±0.000.21±0.620.00±0.001.95±1.77n22222221212121212121200086.7±19.781.57±14.31557.51±142.720.40±0.710.19±0.510.00±0.000.00±0.000.21±0.340.00±0.005.18±15.30n22222222222222222222A: without hook, B: banana-like
head, C: amorphous, D: folded in midpiece, E: others; HRS: homogenization-resistant
spermatids;n: number of males per group.


#### 3.2.2. Offspring Development

Reproductive
performance for theF0 generation was initiated with 26
mating pairs. While not statistically significant, 2, 2, and 1 pairs in the
200, 1000, and 2000 mg/kg/day dose groups, respectively, did not copulate. In
the copulating pairs, nonpregnant females were observed in 1, 2, and 3 cases of
the control, 1000, and 2000 mg/kg/day groups, respectively; again, this did not
result in a significant difference in the fertility index. The F0 breeding resulted in 25, 24, 22, and 22 dams in the control, 200, 1000, and
2000 mg/kg/day dose groups, respectively. The number of litters delivered
resulted in 24, 23, 22, and 22 litters/dose in the control, 200, 1000, and 2000 mg/kg/day dose groups, respectively (see Table 10). One complete litter in the
1000 mg/kg/day dose group was lost on PND
2.Table 10
Litter size and viability index of theF1 generation
rat pups exposed to SE5-OH during gestation and through lactation of the F0 dams.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#7.6±2.7247.3±2.5237.4±2.0226.7±1.922Females7.0±2.0247.3±2.6236.5±1.7227.1±2.322Total14.6±2.82414.7±1.92314.2±1.82213.8±2.322(M/F)(183/168)(169/169)(162/144)(147/157)Number of live offspring at birthMales7.5±2.8247.3±2.6237.2±2.3226.6±2.022Females6.9±2.0247.2±2.4236.4±2.2227.1±2.322Total14.4±3.02414.4±1.62313.6±3.32213.8±2.322(M/F)(179/166)(167/165)(159/140)(146/157)Number of live offspring on PND
4 before cullingMales7.4±2.8247.2±2.6237.1±2.4226.5±1.822Females6.7±2.0247.1±2.3236.2±2.1227.0±2.322Total14.1±2.92414.3±1.62313.3±3.52213.5±2.222(M/F)(178/160)(166/164)(156/137)(144/154)Number of live offspring on PND
4 after cullingMales3.9±0.9244.0±0.2234.0±0.2214.0±0.222Females3.9±0.4244.0±0.2234.0±0.2214.0±0.322Total7.8±1.2248.0±0.0238.0±0.0218.0±0.222Number of live offspring on PND 21Males3.9±0.9244.0±0.2234.0±0.4213.9±0.322Females3.9±0.4244.0±0.2234.0±0.2213.9±0.422Total7.8±1.2248.0±0.0237.9±0.3217.8±0.522ViabilityindexA (%)PND  096.86±10.272498.50±4.992395.78±19.812299.62±1.7722PND  498.23±3.702499.42±1.922393.75±21.382298.58±4.2822PND  21100.00±0.0024100.00±0.002398.81±3.762198.30±5.8422#Mean ± standard deviation; n: number of litters;  PND: postnatal
day; viabilityA index: PND 0: (number
of live offspring born alive/number of offspring born), PND  4: (number of
offspring alive on PND 4/number of offspring born
alive), PND  21: (number
of live weanlings/number of live offspring after culling).ForF1 rat pups, one male and one
female were randomly selected from each litter on PND 21 and were used as breeding animals in the F1 generation
(siblings not paired). One male pup in the control group died on PND 21, and one female 1000 mg/kg/day
pup died on PND 22. In addition,
two F1 females in the control group and one F1 male in
the 1000 mg/kg group that had no mating partner were not subjected to mating.
This resulted in 22, 23, 20, and 22 breeding F1 pairs in the control,
200, 1000, and 2000 mg/kg/day dose groups, respectively. All mating pairs copulated
except for one pair in each of the control and 200 mg/kg/day groups. Three, 3,
1, and 1, nonpregnant females occurred in control, 200, 1000, and 2000 mg/kg/day dose groups, respectively. One female died with vaginal hemorrhage on
GD 23 in the 200 mg/kg/day dose group, which was not considered to be test
article-related. One male of the control group could not fertilize the mating
female, and one male of the 2000 mg/kg/day dose group exhibited unilateral
small and soft testis and small epididymis, and the mated female of this male
had only one implantation with no delivery. These effects were not significant
and not considered to be related to test article administration, as they were
not dose-related responses. Of the dose groups, 18, 18, 19, and 21 F1 dams gave birth to live F2 offspring (see Table 11). There were no
changes attributable to the test article in parturition or nursing. Total
litter loss was observed for one dam each in the 200 and 2000 mg/kg/day dose
groups on PND 4 and PND 1, respectively; however, this was
judged to be incidental as this was not a dose-dependent effect.Table 11
Litter size and viability index of theF2 generation rat pups
exposed to SE5-OH during gestation through lactation.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#8.0±2.8187.9±2.5186.9±1.8186.9±2.920Females7.0±2.4186.6±2.7186.6±2.2185.5±2.720Total15.0±1.91814.4±2.21813.5±2.51812.4±4.220(M/F)(144/126)(142/118)(124/119)(137/109)Number of live offspring at birthMales8.0±2.8187.6±2.6186.9±1.8186.6±3.020Females6.9±2.3186.4±2.5186.6±2.3185.4±2.820Total14.9±1.91814.0±2.31813.4±2.51811.9±4.520(M/F)(144/124)(137/115)(124/118)(131/107)Number of live offspring on PND 4 before cullingMales7.8±2.7187.2±3.1186.4±2.1186.4±3.219Females6.8±2.3186.9±3.0186.3±2.1185.2±3.019Total14.6±1.81813.2±4.01812.7±2.71811.5±5.019(M/F)(140/122)(130/107)(115/113)(127/103)Number of live offspring on PND 4 after cullingMales3.9±0.6184.2±0.7173.9±0.5183.9±1.019Females4.1±0.6183.8±0.7174.0±0.6183.4*±1.019Total8.0±0.0188.0±0.0177.9±0.2187.4*±1.319Number of live offspring on PND 21Males3.8±0.6184.2±0.7173.8±0.6183.9±1.019Females4.1±0.5183.7±0.8174.0±0.6183.4±1.019Total7.9±0.3187.9±0.2177.8±0.5187.4±1.319ViabilityindexA (%)PND  099.23±2.231896.92±4.891899.54±1.961896.21±12.2920PND  497.91±4.151893.61±23.491894.65±12.021894.43±22.3019PND  2198.61±4.041899.26±3.031798.51±4.3418100.00±0.0019#Mean ± standard deviation; n = number of litters; PND: postnatal
day; viabilityA index:  PND 0:  (number of live
offspring born alive/number of offspring born), PND 4: (number
of offspring alive on PND  4/number of offspring born alive), PND 21: (number
of live weanlings/number of live offspring after culling).In theF2 generation, the number of female offspring was reduced at the 2000 mg/kg/day dose on PND 4 (P<.05), but neither the number of live offspring on PND 21 nor the viability index was
affected. This reduction was not
caused by prenatal death or a difference
in the birth index, but was only a reflection of a nonsignificant reduction
in the number of implantations.
The
total litter loss on PND 1 was judged to be incidental, as there was no dose dependency
in the incidence and no abnormalities in the maternal behavior in the dams. There were no adverse effects
observed in any treatment group of F1 and F2 offspring:
clinical signs, body weight, external anomalies, sex ratios, viability index,
physical development, reflex function, organ weight, necropsy findings, and
sexual maturation (data not shown), or viability index (see Table 11).


## 3.1. Developmental Toxicity Study

### 3.1.1. Effects on Dams

With the exception of a transient lack of
weight gain (see Figure2), no abnormal
clinical signs were observed in any of the dams. This included observations for
death, moribundity, abortion, or premature delivery (data not shown). In dams
exposed to 2000 mg/kg, significant decreases in body weight gain were noted on
GD 8–16 but these
values had returned to control levels by GD 20. The same transient lack of
weight gain was noted for dams in the 1000 mg/kg exposure group, but only on GD
8. No treatment-related effects were seen for food consumption, gravid uterine
weights, corrected body weight, and corrected body weight gain; and there were
no abnormalities seen at necropsy (data not shown).Figure 2
Changes in body weight
gain (grams) observed in dams exposed to SE5-OH during gestational days 0–20 during the
developmental toxicity study in rats; significant when compared to control:P*<.05; P**<.01


### 3.1.2. Effects on Embryo-Fetal Development

In all treated groups, there were no
significant differences in number of corpora lutea, implantations, dead
embryos, postimplantation losses, live fetuses, fetal deaths, fetal weight, or sex ratio. Several
isolated embryo-fetal effects were noted during the placental observations, and
external, visceral, and skeletal examinations. These changes are summarized in Table2.Table 2
Isolated embryo-fetal effects noted during
necropsy observations/examinations in the developmental toxicity study in rats.
EffectOccurrenceFusion
of placentaeOccurred in one dam in the 200 mg/kg dose group. No other
placental abnormalities identified in any group.Rudimentary
tailOccurred in one fetus of the 1000 mg/kg dose group. No
other external fetal abnormalities identified in any group.Visceral
anomaly: ventricular septal
defectOccurred in one control group fetus and one 2000 mg/kg
group fetus. No other anomalies identified in any group.Visceral variants: thymic neck remnant, supernumerary
coronary ostium, left umbilical artery, and renal pelvis dilationOccurred
with equal incidence in the control and 2000 mg/kg groups.Skeletal
anomalies/malformations: wavy and nodulated ribsOccurred in one 2000 mg/kg group fetus. No other anomalies
identified in any group.Skeletal variants: foramen hypoglossi double, closure of
transverse foramen of one or more cervical vertebral arches, transverse foramen opening in 7th
cervical arch, splitting of ossification centers in thoracic vertebral
bodies, hemicentric thoracic centrum, dumbbell ossification of thoracic
centrum, lumbarization, cervical ribs, short supernumerary 14th rib, reduced
13th ribs, splitting of sternebraeIncidence
of dumbbell ossification of thoracic centrum was significantly low in the
2000 mg/kg dosing group but was judged incidental with no toxicological
significance.


## 3.1.1. Effects on Dams

With the exception of a transient lack of
weight gain (see Figure2), no abnormal
clinical signs were observed in any of the dams. This included observations for
death, moribundity, abortion, or premature delivery (data not shown). In dams
exposed to 2000 mg/kg, significant decreases in body weight gain were noted on
GD 8–16 but these
values had returned to control levels by GD 20. The same transient lack of
weight gain was noted for dams in the 1000 mg/kg exposure group, but only on GD
8. No treatment-related effects were seen for food consumption, gravid uterine
weights, corrected body weight, and corrected body weight gain; and there were
no abnormalities seen at necropsy (data not shown).Figure 2
Changes in body weight
gain (grams) observed in dams exposed to SE5-OH during gestational days 0–20 during the
developmental toxicity study in rats; significant when compared to control:P*<.05; P**<.01


## 3.1.2. Effects on Embryo-Fetal Development

In all treated groups, there were no
significant differences in number of corpora lutea, implantations, dead
embryos, postimplantation losses, live fetuses, fetal deaths, fetal weight, or sex ratio. Several
isolated embryo-fetal effects were noted during the placental observations, and
external, visceral, and skeletal examinations. These changes are summarized in Table2.Table 2
Isolated embryo-fetal effects noted during
necropsy observations/examinations in the developmental toxicity study in rats.
EffectOccurrenceFusion
of placentaeOccurred in one dam in the 200 mg/kg dose group. No other
placental abnormalities identified in any group.Rudimentary
tailOccurred in one fetus of the 1000 mg/kg dose group. No
other external fetal abnormalities identified in any group.Visceral
anomaly: ventricular septal
defectOccurred in one control group fetus and one 2000 mg/kg
group fetus. No other anomalies identified in any group.Visceral variants: thymic neck remnant, supernumerary
coronary ostium, left umbilical artery, and renal pelvis dilationOccurred
with equal incidence in the control and 2000 mg/kg groups.Skeletal
anomalies/malformations: wavy and nodulated ribsOccurred in one 2000 mg/kg group fetus. No other anomalies
identified in any group.Skeletal variants: foramen hypoglossi double, closure of
transverse foramen of one or more cervical vertebral arches, transverse foramen opening in 7th
cervical arch, splitting of ossification centers in thoracic vertebral
bodies, hemicentric thoracic centrum, dumbbell ossification of thoracic
centrum, lumbarization, cervical ribs, short supernumerary 14th rib, reduced
13th ribs, splitting of sternebraeIncidence
of dumbbell ossification of thoracic centrum was significantly low in the
2000 mg/kg dosing group but was judged incidental with no toxicological
significance.


## 3.2. Reproductive Toxicity Study

### 3.2.1. Parental Animals

Transient
decrease of weight gain and reduced food intake in theF0 and F1 females of 2000 mg/kg/day, and histological change of adrenal cortex (a
diffuse increase in lipid droplets in the zona glomerulosa) in the F0 females of 2000 mg/kg/day were noted (see Table 3). Food intake was reduced in the F0 females in the 1000 mg/kg/day group. In addition, increased liver weight in the F0 female of 1000 and
2000 mg/kg/day dose groups, increased adrenal weight in the F0 males
at the 2000 mg/kg/day dose group, as well as increased thyroid weight in the F1 males (see Table 4) was noted. No adverse clinical signs
(including death, moribundity, abortion, or premature delivery), necropsy findings, or organ weights
were observed in any of the F0 and F1 parental animals.Table 3
Body weight and food consumption in male and female Sprague-Dawley rats during the
two-generation reproductive toxicity study.
GenerationF0F1Dose (mg/kg/day)GenderPeriod (days)020010002000020010002000Body weight (g)MalePremating0195.9±6.6196.5±5.7196.2±6.3195.5±6.259.6±4.9#59.7±5.059.4±5.757.1±5.170540.0±47.2560.1±56.6534.3±46.4539.3±41.2451.8±35.1461.±32.1442.8±37.8445.5±34.9(n=26)(n=26)(n=26)(n=26)(n=23)(n=23)(n=21)(n=22)FemalePremating0152.2±8.5152.5±6.9151.0±6.9151.2±6.855.4±6.057.0±5.857.6±4.854.8±5.570303.9±35.6298.3±21.5289.2±22.4283.0±19.1*266.6±22.9270.0±26.8263.2±21.0252.9±17.1(n=26)(n=26)(n=26)(n=26)(n=24)(n=23)(n=20)(n=22)Gestation7337.9±28.8333.9±21.3323.3±22.7318.5±21*358.9±32.3367.1±34.9352.6±29.6334.7±20.7*14371.0±29.5364.0±23.3355.4±23.8349.3±23.4*395.4±34.4400.9±35.5383.4±29.6367.6±22.9*20443.0±36.4440.2±25.4434.7±26.7428.9±23.6474.9±40.5473.3±39.1453.9±38.9437.3±34.9**(n=25)(n=24)(n=22)(n=22)(n=18)(n=19)(n=19)(n=21)Lactation21355.0±24.2345.8±20.4345.3±21.7343.0±19.5365.3±25.6370.8±28.0368.0±22.8355.8±14.8(n=24)(n=23)(n=21)(n=22)(n=18)(n=17)(n=18)(n=19)Body weight gain (g)MalePremating0–70344.1±45.1363.6±54.4338.1±43.7343.8±40.0392.3±34.1401.8±30.2383.4±34.1388.4±32.1FemalePremating0–70151.7±31.1145.9±20.4138.2±19.3131.9±16.5*211.2±20.8213.0±25.0205.4±19.3198.1±16.9Gestation0–733.2±7.131.3±5.529.6±8.930.7±7.936.8±7.734.6±8.732.1±9.333.3±7.90–1466.3±12.761.4±8.861.7±8.061.5±12.473.2±8.468.4±12.162.8±10.7*66.2±11.80–20138.3±22.3137.6±16.4141.0±11.0141.0±18.5152.8±13.0140.8±24.5133.3±20.8**136.0±26.5Lactation0–211.6±20.3−2.4±20.26.8±20.83.4±13.9−20.3±20.5−17.4±25.4−7.7±23.4−5.5±23.0Food consumption (g)MalePremating7026.2±1.926.9±1.825.4±1.625.1±1.830.5±2.031.3±1.829.0±1.828.9±1.9FemalePremating7019.0±1.718.5±1.617.6±1.2*16.3±0.9**20.3±2.120.6±1.519.2±1.317.5±1.1**Gestation722.6±1.722.1±1.920.7±2.1**20.4±2.4**24.4±1.924.3±2.922.6±3.121.4±2.1**1424.8±2.424.2±2.223.1±2.0*22.8±2.3**26.3±2.226.3±2.824.7±2.623.7±2.6**2026.0±2.425.7±2.626.0±2.225.1±2.127.7±2.126.3±2.525.8±2.126.4±2.6Lactation2168.4±8.568.8±3.769.4±4.067.4±5.467.2±6.667.9±5.364.1±6.465.7±9.2Mean ± standard
deviation; significantly different from control:P<.05*, P<.01**; # the start of dosing for the
F0
groups
was at six weeks of age. The start of dosing for the
F1
group was on day 21
postparturition, to maintain SE5-OH ingestion via lactation; n: number of male and female rats per dose
group.Table 4
Organ weight changes in male and female breeding Sprague-Dawley rats in theF0 and F1 generations of the two-generation rat study.
GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=22)200 (n=22)1000 (n=20)2000 (n=26)MaleThyroids,
absolute organ weight(mg)A30.01±5.45C31.65±5.9429.94±3.8830.32±3.9530.19±4.4831.89±4.9135.15±5.27**32.09±4.18Thyroids,
relative organ weight(mg)B5.23±0.685.33±0.865.33±0.735.34±0.784.55±0.614.76±0.735.48±0.90**4.94±0.58Adrenals, absolute organ weight (mg)61.81±7.0563.95±10.1659.48±7.4068.90±9.01**62.18±7.3063.96±5.8357.61±7.5262.28±9.25Adrenals, relative
organ weight (mg)10.88±1.3110.82±1.6810.57±1.2812.10±1.58**9.41±1.149.56±1.078.95±1.089.62±1.57Seminal Vesicles, absolute organ weight
(g)3.11±0.332.90±0.373.12±0.373.05±0.343.40±0.293.26±0.443.23±0.393.13±0.37Seminal
vesicles, relative organ weight (g)0.55±0.070.49±0.09*0.55±0.080.54±0.060.52±0.080.49±0.010.51±0.080.48±0.06GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=18)200 (n=17)1000 (n=18)2000 (n=19)FemaleLiver,
absolute organ weight (g)13.70±1.3013.99±1.0814.39±1.6214.48±1.1115.01±1.2815.45±1.8616.02±1.4615.71±1.35Liver,
relative organ weight (g)3.86±0.324.05±0.314.17±0.35**4.23±0.29**4.12±0.344.17±0.414.36±0.334.42±0.33*Significantly different from control:P<.05*,
P<.01**; absoluteA organ weight is the weight of the
organ; relativeB organ weight is the weight of the organ relative
to the body weight at necropsy (body weight ratio); meanC ± standard deviation; n:
number of male and female rats per dose group.There were no adverse reproductive effects observed in any treatment group ofF0 and F1 animals
in terms of estrous cycle, copulation
index, fertility index, or precoital period (see Tables 5 and 6). No
adverse effects were seen in gestation period, gestation index, or parturition. No effect on implantation
number was found in the F0 generation although the number of
implantations was slightly, but
significantly, reduced in the F1 dams at 2000 mg/kg/day, there were no effects on implantation in the 200 and 1000 mg/kg/da F1 generation (see Tables 7
and 8). The decrease in
implantation number in the high dose did not significantly affect the birth
index or the number of offspring born alive. SE5-OH administration to male rats
did not affect any of the sperm parameters evaluated, which included sperm
motility, percentage and type of abnormal sperm, tailless sperm, sperm count,
or the number of homogenization-resistant sperm (see Table 9).Table 5
Effect
of SE5-OH administration on the reproductive performance of theF0 generation
rat dams.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.26±0.43#0.0±0.02.5±1.8100.096.2(n=25)1/26(n=26)(n=26)(26/26)(25/26)2004.18±0.320.0±0.02.8±1.692.3100.0(n=24)2/26(n=26)(n=24)(24/26)(24/24)10004.21±0.400.0±0.02.9±2.792.391.7(n=26)0/26(n=26)(n=24)(24/26)(22/24)20004.15±0.380.0±0.03.3±2.596.288.0(n=24)2/26(n=26)(n=25)(25/26)(22/25)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 6
Effect of SE5-OH
administration on theF1 generation rat dam reproductive performance.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.36±0.46#0.1±0.63.1±2.895.585.7(n=24)0/24(n=22)(n=21)(21/22)(18/21)2004.31±0.450.2±0.72.8±1.895.786.4(n=21)2/23(n=23)(n=22)(22/23)(19/22)10004.39±0.410.1±0.22.3±1.3100.095.0(n=20)0/20(n=20)(n=20)(20/20)(19/20)20004.30±0.430.0±0.02.5±1.5100.095.5(n=21)1/22(n=22)(n=22)(22/22)(21/22)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 7
Effects of SE5-OH administration onF0 generation rat dam gestation
and delivery parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.3±0.515.0±3.913.8±4.187.12±21.6496n2425252520022.2±0.415.1±3.613.8±3.489.03±20.4895.8n23242424100022.3±0.615.3±1.713.6±3.389.27±19.68100.0n22222222200022.0±0.314.7±2.113.8±2.393.63±9.28100.0n22222222Significantly different from control:P<.05*,
P<.01**; birthA index = (number of offspring born
alive divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 8
Effects of SE5-OH
administration onF1 generation rat dam gestation and delivery
parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.2±0.415.8±1.214.9±1.993.8±7.4100n=18n=18n=18n=1820022.2±0.415.8±2.514.0±2.388.9±9.2100n=18n=18n=18n=18100022.2±0.413.8±4.112.7±3.988.1±22.894.7n=18n=19n=19n=19200022.3±0.612.6±4.7*11.3±5.184.5±24.895.2n=20n=21n=21n=21BirthA index = (number of offspring born alive
divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 9
Sperm parameters
in control and high-dose male rats used as breeding males in theF0 and
F1 two-generation reproduction study.
Dose (mg/kg/day)Sperm motility (%)HRS (×106/g)Sperm count (cauda epididymal) (×106/g)Abnormal sperm (%)Types of abnormal sperm (%) no.Tailless sperm (%)ABCDEF0 generation091.0±4.583.73±12.52534.73±72.790.81±1.260.25±0.620.04±0.140.00±0.000.42±0.760.10±0.401.96±2.03n26262626262626262626200091.3±3.283.64±10.21537.29±66.920.33±0.370.04±0.140.00±0.000.00±0.000.29±0.350.00±0.001.81±2.83n26262626262626262626F1 generation086.0±20.175.98±17.86551.21±151.940.52±0.830.31±0.430.00±0.000.00±0.000.21±0.620.00±0.001.95±1.77n22222221212121212121200086.7±19.781.57±14.31557.51±142.720.40±0.710.19±0.510.00±0.000.00±0.000.21±0.340.00±0.005.18±15.30n22222222222222222222A: without hook, B: banana-like
head, C: amorphous, D: folded in midpiece, E: others; HRS: homogenization-resistant
spermatids;n: number of males per group.


### 3.2.2. Offspring Development

Reproductive
performance for theF0 generation was initiated with 26
mating pairs. While not statistically significant, 2, 2, and 1 pairs in the
200, 1000, and 2000 mg/kg/day dose groups, respectively, did not copulate. In
the copulating pairs, nonpregnant females were observed in 1, 2, and 3 cases of
the control, 1000, and 2000 mg/kg/day groups, respectively; again, this did not
result in a significant difference in the fertility index. The F0 breeding resulted in 25, 24, 22, and 22 dams in the control, 200, 1000, and
2000 mg/kg/day dose groups, respectively. The number of litters delivered
resulted in 24, 23, 22, and 22 litters/dose in the control, 200, 1000, and 2000 mg/kg/day dose groups, respectively (see Table 10). One complete litter in the
1000 mg/kg/day dose group was lost on PND
2.Table 10
Litter size and viability index of theF1 generation
rat pups exposed to SE5-OH during gestation and through lactation of the F0 dams.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#7.6±2.7247.3±2.5237.4±2.0226.7±1.922Females7.0±2.0247.3±2.6236.5±1.7227.1±2.322Total14.6±2.82414.7±1.92314.2±1.82213.8±2.322(M/F)(183/168)(169/169)(162/144)(147/157)Number of live offspring at birthMales7.5±2.8247.3±2.6237.2±2.3226.6±2.022Females6.9±2.0247.2±2.4236.4±2.2227.1±2.322Total14.4±3.02414.4±1.62313.6±3.32213.8±2.322(M/F)(179/166)(167/165)(159/140)(146/157)Number of live offspring on PND
4 before cullingMales7.4±2.8247.2±2.6237.1±2.4226.5±1.822Females6.7±2.0247.1±2.3236.2±2.1227.0±2.322Total14.1±2.92414.3±1.62313.3±3.52213.5±2.222(M/F)(178/160)(166/164)(156/137)(144/154)Number of live offspring on PND
4 after cullingMales3.9±0.9244.0±0.2234.0±0.2214.0±0.222Females3.9±0.4244.0±0.2234.0±0.2214.0±0.322Total7.8±1.2248.0±0.0238.0±0.0218.0±0.222Number of live offspring on PND 21Males3.9±0.9244.0±0.2234.0±0.4213.9±0.322Females3.9±0.4244.0±0.2234.0±0.2213.9±0.422Total7.8±1.2248.0±0.0237.9±0.3217.8±0.522ViabilityindexA (%)PND  096.86±10.272498.50±4.992395.78±19.812299.62±1.7722PND  498.23±3.702499.42±1.922393.75±21.382298.58±4.2822PND  21100.00±0.0024100.00±0.002398.81±3.762198.30±5.8422#Mean ± standard deviation; n: number of litters;  PND: postnatal
day; viabilityA index: PND 0: (number
of live offspring born alive/number of offspring born), PND  4: (number of
offspring alive on PND 4/number of offspring born
alive), PND  21: (number
of live weanlings/number of live offspring after culling).ForF1 rat pups, one male and one
female were randomly selected from each litter on PND 21 and were used as breeding animals in the F1 generation
(siblings not paired). One male pup in the control group died on PND 21, and one female 1000 mg/kg/day
pup died on PND 22. In addition,
two F1 females in the control group and one F1 male in
the 1000 mg/kg group that had no mating partner were not subjected to mating.
This resulted in 22, 23, 20, and 22 breeding F1 pairs in the control,
200, 1000, and 2000 mg/kg/day dose groups, respectively. All mating pairs copulated
except for one pair in each of the control and 200 mg/kg/day groups. Three, 3,
1, and 1, nonpregnant females occurred in control, 200, 1000, and 2000 mg/kg/day dose groups, respectively. One female died with vaginal hemorrhage on
GD 23 in the 200 mg/kg/day dose group, which was not considered to be test
article-related. One male of the control group could not fertilize the mating
female, and one male of the 2000 mg/kg/day dose group exhibited unilateral
small and soft testis and small epididymis, and the mated female of this male
had only one implantation with no delivery. These effects were not significant
and not considered to be related to test article administration, as they were
not dose-related responses. Of the dose groups, 18, 18, 19, and 21 F1 dams gave birth to live F2 offspring (see Table 11). There were no
changes attributable to the test article in parturition or nursing. Total
litter loss was observed for one dam each in the 200 and 2000 mg/kg/day dose
groups on PND 4 and PND 1, respectively; however, this was
judged to be incidental as this was not a dose-dependent effect.Table 11
Litter size and viability index of theF2 generation rat pups
exposed to SE5-OH during gestation through lactation.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#8.0±2.8187.9±2.5186.9±1.8186.9±2.920Females7.0±2.4186.6±2.7186.6±2.2185.5±2.720Total15.0±1.91814.4±2.21813.5±2.51812.4±4.220(M/F)(144/126)(142/118)(124/119)(137/109)Number of live offspring at birthMales8.0±2.8187.6±2.6186.9±1.8186.6±3.020Females6.9±2.3186.4±2.5186.6±2.3185.4±2.820Total14.9±1.91814.0±2.31813.4±2.51811.9±4.520(M/F)(144/124)(137/115)(124/118)(131/107)Number of live offspring on PND 4 before cullingMales7.8±2.7187.2±3.1186.4±2.1186.4±3.219Females6.8±2.3186.9±3.0186.3±2.1185.2±3.019Total14.6±1.81813.2±4.01812.7±2.71811.5±5.019(M/F)(140/122)(130/107)(115/113)(127/103)Number of live offspring on PND 4 after cullingMales3.9±0.6184.2±0.7173.9±0.5183.9±1.019Females4.1±0.6183.8±0.7174.0±0.6183.4*±1.019Total8.0±0.0188.0±0.0177.9±0.2187.4*±1.319Number of live offspring on PND 21Males3.8±0.6184.2±0.7173.8±0.6183.9±1.019Females4.1±0.5183.7±0.8174.0±0.6183.4±1.019Total7.9±0.3187.9±0.2177.8±0.5187.4±1.319ViabilityindexA (%)PND  099.23±2.231896.92±4.891899.54±1.961896.21±12.2920PND  497.91±4.151893.61±23.491894.65±12.021894.43±22.3019PND  2198.61±4.041899.26±3.031798.51±4.3418100.00±0.0019#Mean ± standard deviation; n = number of litters; PND: postnatal
day; viabilityA index:  PND 0:  (number of live
offspring born alive/number of offspring born), PND 4: (number
of offspring alive on PND  4/number of offspring born alive), PND 21: (number
of live weanlings/number of live offspring after culling).In theF2 generation, the number of female offspring was reduced at the 2000 mg/kg/day dose on PND 4 (P<.05), but neither the number of live offspring on PND 21 nor the viability index was
affected. This reduction was not
caused by prenatal death or a difference
in the birth index, but was only a reflection of a nonsignificant reduction
in the number of implantations.
The
total litter loss on PND 1 was judged to be incidental, as there was no dose dependency
in the incidence and no abnormalities in the maternal behavior in the dams. There were no adverse effects
observed in any treatment group of F1 and F2 offspring:
clinical signs, body weight, external anomalies, sex ratios, viability index,
physical development, reflex function, organ weight, necropsy findings, and
sexual maturation (data not shown), or viability index (see Table 11).


## 3.2.1. Parental Animals

Transient
decrease of weight gain and reduced food intake in theF0 and F1 females of 2000 mg/kg/day, and histological change of adrenal cortex (a
diffuse increase in lipid droplets in the zona glomerulosa) in the F0 females of 2000 mg/kg/day were noted (see Table 3). Food intake was reduced in the F0 females in the 1000 mg/kg/day group. In addition, increased liver weight in the F0 female of 1000 and
2000 mg/kg/day dose groups, increased adrenal weight in the F0 males
at the 2000 mg/kg/day dose group, as well as increased thyroid weight in the F1 males (see Table 4) was noted. No adverse clinical signs
(including death, moribundity, abortion, or premature delivery), necropsy findings, or organ weights
were observed in any of the F0 and F1 parental animals.Table 3
Body weight and food consumption in male and female Sprague-Dawley rats during the
two-generation reproductive toxicity study.
GenerationF0F1Dose (mg/kg/day)GenderPeriod (days)020010002000020010002000Body weight (g)MalePremating0195.9±6.6196.5±5.7196.2±6.3195.5±6.259.6±4.9#59.7±5.059.4±5.757.1±5.170540.0±47.2560.1±56.6534.3±46.4539.3±41.2451.8±35.1461.±32.1442.8±37.8445.5±34.9(n=26)(n=26)(n=26)(n=26)(n=23)(n=23)(n=21)(n=22)FemalePremating0152.2±8.5152.5±6.9151.0±6.9151.2±6.855.4±6.057.0±5.857.6±4.854.8±5.570303.9±35.6298.3±21.5289.2±22.4283.0±19.1*266.6±22.9270.0±26.8263.2±21.0252.9±17.1(n=26)(n=26)(n=26)(n=26)(n=24)(n=23)(n=20)(n=22)Gestation7337.9±28.8333.9±21.3323.3±22.7318.5±21*358.9±32.3367.1±34.9352.6±29.6334.7±20.7*14371.0±29.5364.0±23.3355.4±23.8349.3±23.4*395.4±34.4400.9±35.5383.4±29.6367.6±22.9*20443.0±36.4440.2±25.4434.7±26.7428.9±23.6474.9±40.5473.3±39.1453.9±38.9437.3±34.9**(n=25)(n=24)(n=22)(n=22)(n=18)(n=19)(n=19)(n=21)Lactation21355.0±24.2345.8±20.4345.3±21.7343.0±19.5365.3±25.6370.8±28.0368.0±22.8355.8±14.8(n=24)(n=23)(n=21)(n=22)(n=18)(n=17)(n=18)(n=19)Body weight gain (g)MalePremating0–70344.1±45.1363.6±54.4338.1±43.7343.8±40.0392.3±34.1401.8±30.2383.4±34.1388.4±32.1FemalePremating0–70151.7±31.1145.9±20.4138.2±19.3131.9±16.5*211.2±20.8213.0±25.0205.4±19.3198.1±16.9Gestation0–733.2±7.131.3±5.529.6±8.930.7±7.936.8±7.734.6±8.732.1±9.333.3±7.90–1466.3±12.761.4±8.861.7±8.061.5±12.473.2±8.468.4±12.162.8±10.7*66.2±11.80–20138.3±22.3137.6±16.4141.0±11.0141.0±18.5152.8±13.0140.8±24.5133.3±20.8**136.0±26.5Lactation0–211.6±20.3−2.4±20.26.8±20.83.4±13.9−20.3±20.5−17.4±25.4−7.7±23.4−5.5±23.0Food consumption (g)MalePremating7026.2±1.926.9±1.825.4±1.625.1±1.830.5±2.031.3±1.829.0±1.828.9±1.9FemalePremating7019.0±1.718.5±1.617.6±1.2*16.3±0.9**20.3±2.120.6±1.519.2±1.317.5±1.1**Gestation722.6±1.722.1±1.920.7±2.1**20.4±2.4**24.4±1.924.3±2.922.6±3.121.4±2.1**1424.8±2.424.2±2.223.1±2.0*22.8±2.3**26.3±2.226.3±2.824.7±2.623.7±2.6**2026.0±2.425.7±2.626.0±2.225.1±2.127.7±2.126.3±2.525.8±2.126.4±2.6Lactation2168.4±8.568.8±3.769.4±4.067.4±5.467.2±6.667.9±5.364.1±6.465.7±9.2Mean ± standard
deviation; significantly different from control:P<.05*, P<.01**; # the start of dosing for the
F0
groups
was at six weeks of age. The start of dosing for the
F1
group was on day 21
postparturition, to maintain SE5-OH ingestion via lactation; n: number of male and female rats per dose
group.Table 4
Organ weight changes in male and female breeding Sprague-Dawley rats in theF0 and F1 generations of the two-generation rat study.
GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=22)200 (n=22)1000 (n=20)2000 (n=26)MaleThyroids,
absolute organ weight(mg)A30.01±5.45C31.65±5.9429.94±3.8830.32±3.9530.19±4.4831.89±4.9135.15±5.27**32.09±4.18Thyroids,
relative organ weight(mg)B5.23±0.685.33±0.865.33±0.735.34±0.784.55±0.614.76±0.735.48±0.90**4.94±0.58Adrenals, absolute organ weight (mg)61.81±7.0563.95±10.1659.48±7.4068.90±9.01**62.18±7.3063.96±5.8357.61±7.5262.28±9.25Adrenals, relative
organ weight (mg)10.88±1.3110.82±1.6810.57±1.2812.10±1.58**9.41±1.149.56±1.078.95±1.089.62±1.57Seminal Vesicles, absolute organ weight
(g)3.11±0.332.90±0.373.12±0.373.05±0.343.40±0.293.26±0.443.23±0.393.13±0.37Seminal
vesicles, relative organ weight (g)0.55±0.070.49±0.09*0.55±0.080.54±0.060.52±0.080.49±0.010.51±0.080.48±0.06GenerationF0F1Dose (mg/kg/day) (n)0 (n=26)200 (n=26)1000 (n=26)2000 (n=26)0 (n=18)200 (n=17)1000 (n=18)2000 (n=19)FemaleLiver,
absolute organ weight (g)13.70±1.3013.99±1.0814.39±1.6214.48±1.1115.01±1.2815.45±1.8616.02±1.4615.71±1.35Liver,
relative organ weight (g)3.86±0.324.05±0.314.17±0.35**4.23±0.29**4.12±0.344.17±0.414.36±0.334.42±0.33*Significantly different from control:P<.05*,
P<.01**; absoluteA organ weight is the weight of the
organ; relativeB organ weight is the weight of the organ relative
to the body weight at necropsy (body weight ratio); meanC ± standard deviation; n:
number of male and female rats per dose group.There were no adverse reproductive effects observed in any treatment group ofF0 and F1 animals
in terms of estrous cycle, copulation
index, fertility index, or precoital period (see Tables 5 and 6). No
adverse effects were seen in gestation period, gestation index, or parturition. No effect on implantation
number was found in the F0 generation although the number of
implantations was slightly, but
significantly, reduced in the F1 dams at 2000 mg/kg/day, there were no effects on implantation in the 200 and 1000 mg/kg/da F1 generation (see Tables 7
and 8). The decrease in
implantation number in the high dose did not significantly affect the birth
index or the number of offspring born alive. SE5-OH administration to male rats
did not affect any of the sperm parameters evaluated, which included sperm
motility, percentage and type of abnormal sperm, tailless sperm, sperm count,
or the number of homogenization-resistant sperm (see Table 9).Table 5
Effect
of SE5-OH administration on the reproductive performance of theF0 generation
rat dams.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.26±0.43#0.0±0.02.5±1.8100.096.2(n=25)1/26(n=26)(n=26)(26/26)(25/26)2004.18±0.320.0±0.02.8±1.692.3100.0(n=24)2/26(n=26)(n=24)(24/26)(24/24)10004.21±0.400.0±0.02.9±2.792.391.7(n=26)0/26(n=26)(n=24)(24/26)(22/24)20004.15±0.380.0±0.03.3±2.596.288.0(n=24)2/26(n=26)(n=25)(25/26)(22/25)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 6
Effect of SE5-OH
administration on theF1 generation rat dam reproductive performance.
Dose (mg/kg/day)Mean estrus cycleIncidence of irregular estrus cycleMating periodCopulation
index(%)AFertility index(%)BNumber of estrusDay of conceiving04.36±0.46#0.1±0.63.1±2.895.585.7(n=24)0/24(n=22)(n=21)(21/22)(18/21)2004.31±0.450.2±0.72.8±1.895.786.4(n=21)2/23(n=23)(n=22)(22/23)(19/22)10004.39±0.410.1±0.22.3±1.3100.095.0(n=20)0/20(n=20)(n=20)(20/20)(19/20)20004.30±0.430.0±0.02.5±1.5100.095.5(n=21)1/22(n=22)(n=22)(22/22)(21/22)#Mean ± standard deviation; numberA
of copulated females/number of pairs; numberB of pregnant
females/number of copulated females; n: number of mating pairs.Table 7
Effects of SE5-OH administration onF0 generation rat dam gestation
and delivery parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.3±0.515.0±3.913.8±4.187.12±21.6496n2425252520022.2±0.415.1±3.613.8±3.489.03±20.4895.8n23242424100022.3±0.615.3±1.713.6±3.389.27±19.68100.0n22222222200022.0±0.314.7±2.113.8±2.393.63±9.28100.0n22222222Significantly different from control:P<.05*,
P<.01**; birthA index = (number of offspring born
alive divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 8
Effects of SE5-OH
administration onF1 generation rat dam gestation and delivery
parameters.
Dose (mg/kg/day)Gestation length (days)Number of implantation sitesNumber of offspring born aliveBirthindexA (%)GestationindexB (%)022.2±0.415.8±1.214.9±1.993.8±7.4100n=18n=18n=18n=1820022.2±0.415.8±2.514.0±2.388.9±9.2100n=18n=18n=18n=18100022.2±0.413.8±4.112.7±3.988.1±22.894.7n=18n=19n=19n=19200022.3±0.612.6±4.7*11.3±5.184.5±24.895.2n=20n=21n=21n=21BirthA index = (number of offspring born alive
divided by the number of implantations); gestationB index =
(number of females with live offspring divided by the number of pregnant
females); n: number of dams per group.Table 9
Sperm parameters
in control and high-dose male rats used as breeding males in theF0 and
F1 two-generation reproduction study.
Dose (mg/kg/day)Sperm motility (%)HRS (×106/g)Sperm count (cauda epididymal) (×106/g)Abnormal sperm (%)Types of abnormal sperm (%) no.Tailless sperm (%)ABCDEF0 generation091.0±4.583.73±12.52534.73±72.790.81±1.260.25±0.620.04±0.140.00±0.000.42±0.760.10±0.401.96±2.03n26262626262626262626200091.3±3.283.64±10.21537.29±66.920.33±0.370.04±0.140.00±0.000.00±0.000.29±0.350.00±0.001.81±2.83n26262626262626262626F1 generation086.0±20.175.98±17.86551.21±151.940.52±0.830.31±0.430.00±0.000.00±0.000.21±0.620.00±0.001.95±1.77n22222221212121212121200086.7±19.781.57±14.31557.51±142.720.40±0.710.19±0.510.00±0.000.00±0.000.21±0.340.00±0.005.18±15.30n22222222222222222222A: without hook, B: banana-like
head, C: amorphous, D: folded in midpiece, E: others; HRS: homogenization-resistant
spermatids;n: number of males per group.


## 3.2.2. Offspring Development

Reproductive
performance for theF0 generation was initiated with 26
mating pairs. While not statistically significant, 2, 2, and 1 pairs in the
200, 1000, and 2000 mg/kg/day dose groups, respectively, did not copulate. In
the copulating pairs, nonpregnant females were observed in 1, 2, and 3 cases of
the control, 1000, and 2000 mg/kg/day groups, respectively; again, this did not
result in a significant difference in the fertility index. The F0 breeding resulted in 25, 24, 22, and 22 dams in the control, 200, 1000, and
2000 mg/kg/day dose groups, respectively. The number of litters delivered
resulted in 24, 23, 22, and 22 litters/dose in the control, 200, 1000, and 2000 mg/kg/day dose groups, respectively (see Table 10). One complete litter in the
1000 mg/kg/day dose group was lost on PND
2.Table 10
Litter size and viability index of theF1 generation
rat pups exposed to SE5-OH during gestation and through lactation of the F0 dams.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#7.6±2.7247.3±2.5237.4±2.0226.7±1.922Females7.0±2.0247.3±2.6236.5±1.7227.1±2.322Total14.6±2.82414.7±1.92314.2±1.82213.8±2.322(M/F)(183/168)(169/169)(162/144)(147/157)Number of live offspring at birthMales7.5±2.8247.3±2.6237.2±2.3226.6±2.022Females6.9±2.0247.2±2.4236.4±2.2227.1±2.322Total14.4±3.02414.4±1.62313.6±3.32213.8±2.322(M/F)(179/166)(167/165)(159/140)(146/157)Number of live offspring on PND
4 before cullingMales7.4±2.8247.2±2.6237.1±2.4226.5±1.822Females6.7±2.0247.1±2.3236.2±2.1227.0±2.322Total14.1±2.92414.3±1.62313.3±3.52213.5±2.222(M/F)(178/160)(166/164)(156/137)(144/154)Number of live offspring on PND
4 after cullingMales3.9±0.9244.0±0.2234.0±0.2214.0±0.222Females3.9±0.4244.0±0.2234.0±0.2214.0±0.322Total7.8±1.2248.0±0.0238.0±0.0218.0±0.222Number of live offspring on PND 21Males3.9±0.9244.0±0.2234.0±0.4213.9±0.322Females3.9±0.4244.0±0.2234.0±0.2213.9±0.422Total7.8±1.2248.0±0.0237.9±0.3217.8±0.522ViabilityindexA (%)PND  096.86±10.272498.50±4.992395.78±19.812299.62±1.7722PND  498.23±3.702499.42±1.922393.75±21.382298.58±4.2822PND  21100.00±0.0024100.00±0.002398.81±3.762198.30±5.8422#Mean ± standard deviation; n: number of litters;  PND: postnatal
day; viabilityA index: PND 0: (number
of live offspring born alive/number of offspring born), PND  4: (number of
offspring alive on PND 4/number of offspring born
alive), PND  21: (number
of live weanlings/number of live offspring after culling).ForF1 rat pups, one male and one
female were randomly selected from each litter on PND 21 and were used as breeding animals in the F1 generation
(siblings not paired). One male pup in the control group died on PND 21, and one female 1000 mg/kg/day
pup died on PND 22. In addition,
two F1 females in the control group and one F1 male in
the 1000 mg/kg group that had no mating partner were not subjected to mating.
This resulted in 22, 23, 20, and 22 breeding F1 pairs in the control,
200, 1000, and 2000 mg/kg/day dose groups, respectively. All mating pairs copulated
except for one pair in each of the control and 200 mg/kg/day groups. Three, 3,
1, and 1, nonpregnant females occurred in control, 200, 1000, and 2000 mg/kg/day dose groups, respectively. One female died with vaginal hemorrhage on
GD 23 in the 200 mg/kg/day dose group, which was not considered to be test
article-related. One male of the control group could not fertilize the mating
female, and one male of the 2000 mg/kg/day dose group exhibited unilateral
small and soft testis and small epididymis, and the mated female of this male
had only one implantation with no delivery. These effects were not significant
and not considered to be related to test article administration, as they were
not dose-related responses. Of the dose groups, 18, 18, 19, and 21 F1 dams gave birth to live F2 offspring (see Table 11). There were no
changes attributable to the test article in parturition or nursing. Total
litter loss was observed for one dam each in the 200 and 2000 mg/kg/day dose
groups on PND 4 and PND 1, respectively; however, this was
judged to be incidental as this was not a dose-dependent effect.Table 11
Litter size and viability index of theF2 generation rat pups
exposed to SE5-OH during gestation through lactation.
Dose (mg/kg/day)0n200n1000n2000nNumber of total offspring at birthMales#8.0±2.8187.9±2.5186.9±1.8186.9±2.920Females7.0±2.4186.6±2.7186.6±2.2185.5±2.720Total15.0±1.91814.4±2.21813.5±2.51812.4±4.220(M/F)(144/126)(142/118)(124/119)(137/109)Number of live offspring at birthMales8.0±2.8187.6±2.6186.9±1.8186.6±3.020Females6.9±2.3186.4±2.5186.6±2.3185.4±2.820Total14.9±1.91814.0±2.31813.4±2.51811.9±4.520(M/F)(144/124)(137/115)(124/118)(131/107)Number of live offspring on PND 4 before cullingMales7.8±2.7187.2±3.1186.4±2.1186.4±3.219Females6.8±2.3186.9±3.0186.3±2.1185.2±3.019Total14.6±1.81813.2±4.01812.7±2.71811.5±5.019(M/F)(140/122)(130/107)(115/113)(127/103)Number of live offspring on PND 4 after cullingMales3.9±0.6184.2±0.7173.9±0.5183.9±1.019Females4.1±0.6183.8±0.7174.0±0.6183.4*±1.019Total8.0±0.0188.0±0.0177.9±0.2187.4*±1.319Number of live offspring on PND 21Males3.8±0.6184.2±0.7173.8±0.6183.9±1.019Females4.1±0.5183.7±0.8174.0±0.6183.4±1.019Total7.9±0.3187.9±0.2177.8±0.5187.4±1.319ViabilityindexA (%)PND  099.23±2.231896.92±4.891899.54±1.961896.21±12.2920PND  497.91±4.151893.61±23.491894.65±12.021894.43±22.3019PND  2198.61±4.041899.26±3.031798.51±4.3418100.00±0.0019#Mean ± standard deviation; n = number of litters; PND: postnatal
day; viabilityA index:  PND 0:  (number of live
offspring born alive/number of offspring born), PND 4: (number
of offspring alive on PND  4/number of offspring born alive), PND 21: (number
of live weanlings/number of live offspring after culling).In theF2 generation, the number of female offspring was reduced at the 2000 mg/kg/day dose on PND 4 (P<.05), but neither the number of live offspring on PND 21 nor the viability index was
affected. This reduction was not
caused by prenatal death or a difference
in the birth index, but was only a reflection of a nonsignificant reduction
in the number of implantations.
The
total litter loss on PND 1 was judged to be incidental, as there was no dose dependency
in the incidence and no abnormalities in the maternal behavior in the dams. There were no adverse effects
observed in any treatment group of F1 and F2 offspring:
clinical signs, body weight, external anomalies, sex ratios, viability index,
physical development, reflex function, organ weight, necropsy findings, and
sexual maturation (data not shown), or viability index (see Table 11).


## 4. Discussion

Soy is the most widely used food plant in
the world and has been cultivated for over 4500 years, resulting in human soy
isoflavone consumption for several millennia [29]. Recently published data by the
USDA [30] indicate that daidzein, genistein, and glycitein are constituents in
a wide variety of legumes, prepared foods, spices, teas, and of course,
soy-based foods, including infant formula, tofu, tempeh, cheese, beverages,
noodles, sauces, chips, and meat substitutes. In unfermented food products, the
glycosylated forms (i.e.,
daidzin, genistin, glycitin, and their derivatives) are more abundant than the
aglycone forms. The widespread historical exposure to soy isoflavones in the
diet, without adverse human health effects, and the overall body of available
scientific evidence, is a
compelling evidence of their safety. While average Japanese isoflavone intake
has been estimated at 50 mg/day, western consumption is approximated at 5 mg/day [31, 32].There has been a concern
that ingestion of soy isoflavones may lead to adverse effects on testes and
ovaries [33–36]. Unlike diethylstilbestrol (DES), equol at 1000 μg/animal/day injected subcutaneously for
five days failed to demonstrate estrogenic, or antiestrogenic activity in the
uterine wall of neonatal Sprague-Dawley (SD) rats [37]. Other research found
that equol administration directly to the uterine horn in ovariectomized rats
did not increase uterine weight, and although equol did weakly bind to the
estrogen receptor with a high dissociation rate, it did not demonstrate
antiestrogenic activity [38]. In concert with findings reported by Thompson et al. [38], Wood et al. [39] found that high doses of
genistein and daidzein or equol had minimal uterotrophic or mammotrophic
effects in an ovariectomized monkey (Macaca
fascicularis) model. In the normal postmenopausal mammary gland of
cynomolgus macaques, equol at an equivalent human dose of 105 mg/day for
greater than six months lacked estrogenic effects, and did not promote lesions in the mammary tissue [40]. Rachon et al. [41–43] found that equol in the diet at 400 mg per kg of food (delivering approx.
26.16 mg equol/kg body weight/day to the rat) for three months to
ovariectomized rats significantly increased serum prolactin and lutenizing
hormone levels (P<.05), but had no effect on pituitary estrogen
receptor (ER)-α or -β gene expression.
However, equol administration did increase an isoform of the estrogen receptor
(Terp-1) in the pituitary of these ovariectomized rats, and increased the
number of terminal ducts and type II lobules, compared to control rats. Equol dosed
at 3.68 mg/kg/day in this same study had no effect on rat mammary tissue. In
this same rat model, equol (26.16 mg/kg body weight/day) significantly
increased uterine weights and increased the epithelial height and thickness of
the uterine stroma and myometrium, and significantly increased levels of
uterine insulin-like growth factor 1, progesterone receptor, and complement
protein 3 mRNA, while 3.68 mg equol/kg bodyweight/day had no effect [44]. Subcutaneous
administration of up to 1000 μg equol in rat pups during PND 1–5 or PND 10–14 lowered
uterine weight at later ages, but did not affect ER levels [45]. Equol
administration (approx. 27.48 mg/kg body weight/day) in ovariectomized rats for
six weeks increased uterine weight, decreased weight gain, and attenuated
trabecular bone loss (but not proximal tibia bone loss), while increasing its
density, indicating mild bone sparing effects [46]. These studies indicate that
there is an uncertain degree of difference in species- and organ-specific
sensitivity to equol administration.SE5-OH is a unique fermented soy germ food
product that is intended to deliver equol to individuals who cannot produce it
endogenously. The studies currently described were conducted to determine any
potential developmental and reproductive effects of SE5-OH. In the
developmental study, isolated embryo-fetal effects were noted during necropsy,
which included the fusion of placentae in one dam in the 200 mg/kg/day dose
group. As no other placental abnormalities were identified in this study, this
effect was determined not to be test article-related. One control group fetus
and one high-dose fetus were found to have a ventricular septal defect, and
visceral variants that included a thymic neck remnant, supernumerary coronary
ostium, left umbilical artery, and renal pelvis dilations which occurred with
equal incidence in these dose groups and were determined not to be test
article-related. Dumbell ossification of the thoracic centrum was significantly
lower in the high-dose group, but the overall incidence of the observed
skeletal variants (as indicated in Table4) was determined not to be test
article-related because they are similarly seen in untreated Crl:CD(SD) rats.An increase in relative (but not absolute)
liver weight was noted in theF0 1000 and 2000 mg/kg/day dams, and
in the 2000 mg/kg/day F1 dams, but was not seen in the male rats. The
increase at the 1000 mg/kg/day dose did not exceed the limits of 5%
variability, which is generally considered within the accepted level of variability
to the control rats. The relative liver weight at the 2000 mg/kg/day dose had
greater than 5% variability from controls. There was a significant increase in
the absolute and relative thyroid weight in the 1000 mg/kg/day dose group, but
this was not considered related to SE5-OH treatment, as this effect was not
noted in any other dose levels. The highest dose of SE5-OH (2000 mg/kg/day) had
some indications of toxicity at the gestational and conception stages in the
rat model.The adrenal
cortex ofF0 females at the 2000 mg/kg/day SE5-OH dose had an
increased number of diffuse lipid droplets (4/10 in the high-dose group versus 0/10 in the controls) noted in
the glomerular zone of the adrenal glands.
Lipid droplets are the
intracellular stores of cholesterol esters, precursors of hormones formed in
the rat adrenal glands [47]. It has been documented that cell
enlargement and widening of the zona glomerulosa of the adrenal cortex (associated
with an increase in lipid droplet volume) may be induced via androgen receptor
binding, and that isoflavones (including equol as an antiandrogen [48]) have
been reported to alter androgen receptor expression and adrenocortical function
[49, 50]. However, the exact mechanism is not known, as isoflavones have been
shown to increase or decrease androgen production, depending on the model used [51–54]. Wood et al. [53] noted that
consumption of a soy protein providing 129 mg/day isoflavones (which provided
approx. 31 mg daidzein per day)
by female cynomolgus monkeys for 36 months did not alter serum androgen levels,
but decreased adrenal weight (P<.0006). However, previous postmenopausal oral
contraceptive administration abolished this effect (P<.01). Diffuse
lipid droplets in the adrenal cortex have been noted in the rat model as a
consequence of advancing age [47], and coupled with pregnancy, they may have been a
factor in the results of this study. The maximal plasma concentration was 390±90 and 414±32 ng/mL, respectively,
for the S- and R-enantiomers.Although a significant reduction in the
number of implantations in this study is thought to have occurred due to
lowered caloric intake in the high-dose pregnant dams, it still remains that
there was an overall adverse effect, albeit an indirect one (i.e., an effect of reduced caloric
intake on decreased body weights) and not a product of the intrinsic toxicity
of SE5-OH on the reproductive ability of the rats in this study. Even though a transient decrease in body weight gain was noted inF0 and F1 dams of the 2000 mg/kg/day dose group, no changes were observed in fetal viability, intrauterine growth, and
fetal morphology (external, skeletal, and visceral) that were attributed
to SE5-OH treatment. Isolated
embryo-fetal alterations were noted, but were not consistent with SE5-OH
treatment or significantly different from controls. It is known that food
restriction affects the adult female rat reproductive capacity and may decrease
the number of corpus luteum graviditatis [55, 56]. Equol administration to ovariectomized
rats at approximately 27.48 mg/kg/day for six weeks [44] resulted in decreased
weight gain, intra-abdominal fat, plasma leptin, and total cholesterol levels.
Soy isoflavone administration to male, female, and ovariectomized rats reduced food intake and weekly
body weight gain, while increasing serum isoflavones, including equol [57]. A
decrease in implantations was noted at the 2000 mg/kg/day dose group; however, since no adverse effects were noted on organ
weights or histopathological examination of the reproductive organs, mating ability, fertility, pregnancy, parturition, or nursing behavior in F0 or F1 animals,
this slight decrease in
implantation number was not a substantive or meaningful effect. Further, the
absence of an effect on the fecundity of the high-dose animals in the
reproduction study indicates this decrease in implantation was an anomalous
finding, and is supported by the lack of reproductive toxicity by daidzein in
rats reported by Lamartiniere et al.
[34]. In a previous work [26], no adverse observations were found in adult rats
at up to 2000 mg/kg/day SE5-OH in a subchronic 90-day study. In the present
study, no adverse toxicological observations were made at either the
reproductive or developmental stages in the either sex of the rat model at the
1000 mg/kg/day dose. From the combined studies herein, we can conclude that in
the developmental toxicity phase of the study, a
no-observed-adverse-effect-level (NOAEL) for developmental effects of SE5-OH is
2000 mg/kg/day, based on the lack of significant embryo-to-fetal stage effect.
From the reproductive study, the NOAEL for SE5-OH determined for both male and
female rats is 1000 mg/kg/day (6.5 mg equol/kg/day), based on the reduction of
body weight, implantations, and live births in the F1 and F2 animals at the 2000 mg/kg/day dose level.



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*Source: 307618-2008-12-15.xml*