Food and Chemical Toxicology 40 (2002) 18071813

www.elsevier.com/locate/foodchemtox

Eects of propyl paraben on the male reproductive system

S. Oishi*
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health,
3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan
Accepted 7 July 2002

Abstract
Parabens are p-hydroxybenzoic acid ester compounds widely used as preservatives in foods, cosmetics, toiletries and pharmaceuticals. These compounds exert a weak estrogenic activity as determined by in vitro estrogen receptor assay and in vivo uterotrophic assay. In a previous study, it was demonstrated by the present author that exposure of post-weaning mammals to butyl
paraben adversely aects the secretion of testosterone and the function of the male reproductive system. In the present study, it is
shown that propyl paraben also adversely aects the hormonal secretion and the male reproductive functions. Propyl paraben was
administered to 3-week-old rats which were divided into four groups of eight animals each, at doses of 0.00, 0.01, 0.10 and 1.00%
with the AIN93G modied diet. At the end of 4 weeks, the rats were sacriced by decapitation and the weights of testes, epididymides, prostates, seminal vesicles and preputial glands were determined. There were no treatment-related eects of propyl paraben
on the organ weights in any of the study groups. The cauda epididymal sperm reserves and concentrations decreased in a dosedependent manner and the dierence was signicant at dose of 0.10% and above. Daily sperm production and its eciency in the
testis of all groups receiving propyl paraben signicantly decreased. The serum testosterone concentration decreased in a dosedependent manner and the decrease was signicant in the group that received the highest dose. The exposure level at which this
eect was observed is the same as the upper-limit acceptable daily intake (10 mg/kg body weight/day) of parabens in the European
Community and Japan.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Acceptable daily intake (ADI); Paraben; Preservatives; Propyl paraben; Rat; Sperm; Testosterone

1. Introduction
There are many reports of decreased sperm counts in
men and increased incidence of disorders of the male
reproductive tract (Matlai and Beral, 1985; Sharpe and
Skakkebaek, 1993; Adami et al., 1994; Ginsberg, 1994;
Auger et al., 1995; Pajarinen et al., 1997; Swan et al.,
2000; Moller, 2001). Although contradictory and noteworthy results have been published (Bromwich et al.,
1994; Olsen et al., 1995; Bujan et al., 1996; Vierula et al.,
1996), the reasons for the decline in the quality of semen
and sperm and increased incidence of male reproductive
disorders are subjects of current research. Exposure to
natural or synthetic estrogens may adversely aect
human health, particularly with regard to the reproAbbreviations: DSP, Daily sperm production
* Corresponding author. Tel.: +81-3-3363-3231; fax: +81-3-33684060.
E-mail address: oishi@tokyo-eiken.go.jp (S. Oishi).

ductive cycle and reproductive function. In recent years,

it has been demonstrated that a number of environmental pollutants have activities similar to those of
17-estradiol or antiandrogens (Colborn et al., 1993;
Daston et al., 1997). These include bisphenol A (Nagel
et al., 1997; vom Saal et al., 1998), alkylphenol (White et
al., 1994; Nimrod and Benson, 1996; Routledge and
Sumpter, 1997), PCBs (Bitman and Cecil, 1970; Korach
et al., 1987; Bergeron et al., 1994) and phthalates (Oishi
and Hiraga, 1980; Coldham et al., 1997; Bolger et al.,
1998; Zacharewsky et al., 1998; Nakai et al., 1999).
Parabens are the most commonly used preservatives
in cosmetics, toiletries, pharmaceuticals and foods,
because of their relatively low toxicity in humans and
their eective antimicrobial activity (Elder, 1984). One or
more parabens are found in all types of cosmetic products and are used in more than 13,200 formulations
(Elder, 1984). Parabens were detected in 77% of rinse-o
products and in 99% of leave-on products, and the total
paraben content in paraben-positive cosmetics was

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S. Oishi / Food and Chemical Toxicology 40 (2002) 18071813

0.010.87% (Rastogi et al., 1995). Ethyl, n-propyl,

i-propyl, i-butyl and n-butyl esters of p-hydroxybenzoic
acid are allowed as food additives in Japan (Ministry of
Health and Welfare, Japan, 1994) and the limits of these
esters are regulated as the total amount of p-hydroxybenzoic acid. According to Ishiwata et al. (1999), the
daily intake of parabens from foods is 1.06 mg/person/
day. The maximum levels of parabens in pharmaceutical
products seldom exceed 1% (w/w), and the EEC directive
76/768/EEC and Danish cosmetic regulations permit the
preservation of cosmetic products with methyl paraben,
ethyl paraben, propyl paraben and butyl paraben up to a
maximum combined concentration of 0.8% (w/w). Based
on several estimates, the total paraben exposure is 76 mg/
day or 1.3 mg/kg body weight/day, with food accounting
for approximately 1 mg/day, cosmetics and personal products 50 mg/day and drugs 25 mg/day (Soni et al., 2001).
p-Hydroxy substitution on the aromatic ring has been
recognized as an important requirement for the estrogenic activity of some chemicals such as alkylphenols
(Jordan and Liberman, 1984; Nishihara and Nishikawa,
2001). Parabens are esters of p-hydroxybenzoic acid.
Because of their structural similarity to alkylphenols,
the estrogenic activity of parabens and p-hydroxybenzoic acid, the main metabolite of parabens, was
studied by in vitro recombinant yeast assay, human
estrogen receptor assay, and E-screen (Lemini et al.,
1997; Routledge et al., 1998; Blaier et al., 2000; Hossaiani et al., 2000; Pedersen et al., 2000; Satoh et al.,
2000; Okubo et al., 2001). The magnitude of estrogenic
response increased with the alkyl group size, as shown
by the fact that ethyl paraben, propyl paraben and butyl
paraben are approximately 150,000-, 30,000- and
10,000-fold less potent than 17b-estradiol, respectively.
Propyl paraben was found to be equal in potency to
4-nonylphenol, whereas butyl paraben was found to be
three-fold more potent (Routledge et al., 1998). These in
vitro studies demonstrated that parabens have weak
estrogenicity and butyl ester showed greater activity
than the corresponding methyl, ethyl and propyl esters.
The antispermatogenic activity of butyl paraben was the
same as or more potent than those of alkylphenols in in
vivo rat and mouse studies (Oishi, 2001, 2002). In the
present report, it is show that propyl paraben also
adversely aects the secretion of testosterone and the
functions of the male reproductive system.

2. Materials and methods

2.1. Chemicals
Trypan blue, Triton X-100, diethyl ether and n-propyl-p-hydroxy-benzoate (propyl paraben) of at least
99% purity were obtained Wako Pure Chemical Industries, Ltd (Osaka, Japan). A testosterone enzyme

immunoassay kit was purchased from Oxford Biomedical Research, Inc. (Oxford, MI, USA).
2.2. Animals and treatment
Immature Crj:Wistar rats were purchased from
Charles River Japan, Inc., Kanagawa. The animals were
housed individually in wire-bottomed stainless-steel
cages in a controlled environment with temperature
maintained at 2124  C and relative humidity at
55  5%. A 12-h light/dark cycle was maintained
throughout the experimental period. The rats, aged
1921 days and weighing 52.5  2.17 g, were divided into
four groups of eight animals each. Propyl paraben was
given with the slightly modied AIN93G diet (Reeves et
al., 1993) at 0.00, 0.01, 0.10 and 1.00% for 4 weeks.
Soybean oil in the AIN93G diet was replaced with corn
oil in order to avoid the eect of estrogenic compounds
such as daidzein, genistein and other phytoestrogens, in
soybean. Feed and water were given ad lib. for the
duration of the experiment. Body weights and food
consumption were measured daily. After the 4-week
exposure, the rats were sacriced by decapitation. Blood
samples were collected from each animal and the serum
was separated and stored at 80  C until use in the testosterone assay. The testes, epididymides, ventral prostates, preputial glands and seminal vesicles with
coagulation glands were removed and weighed. The
testes and epididymides were stored at 80  C until
sperm counts were determined.
2.3. Sperm counts in the testis
Daily sperm production (DSP) and its eciency
(DSP/g testis) in the testis were determined as reported
previously (Oishi, 2001). Briey, the testis was thawed,
re-weighed, minced with scissors, and homogenized for
1 min in 50 ml of physiological saline solution containing 0.05% (by volume) Triton X-100, which was performed using a Polytron homogenizer (Kinematica
GmbH, Luzerne, Switzerland) at setting 6. Steps 1719
spermatids (Stages IVVIII) survive the homogenization and their elongated nuclei can then be counted
using a Thoma-type hemocytometer.
2.4. Sperm reserves of cauda epididymides
After thawing the epididymides, the tail regions were
removed (Filler, 1993) and re-weighed before nely
mincing them with ophthalmologic scissors in 5.0 ml of
physiological saline solution. The homogenate was ltered through a nylon mesh and then 0.1 ml of the ltrate was diluted with 3.0 ml of saline solution
containing 4% trypan blue. From this solution, 20-ml
aliquots were placed on the hemocytometer for counting
in the same manner as that for testicular sperm counts.

S. Oishi / Food and Chemical Toxicology 40 (2002) 18071813

2.5. Testosterone measurement

Testosterone in serum was extracted with diethyl
ether and its concentration was measured in duplicate
using an enzyme immunoassay kit. The assay was conducted according to the manufacturers instructions.
2.6. Statistical analyses
The data were rst analyzed by Bartletts test for
homogeneity of variance. If the results were signicant,
the data were analyzed by the KruskalWallis nonparametric analysis of variance (ANOVA) and by
Steels test. If the Bartletts test was not signicant,
ANOVA was conducted. If ANOVA was signicant,
Dunnetts test was performed (Gad and Weil, 1994; Zar,
1999). Trend analysis (dose-dependent analysis) was
performed according to Jonckheere (1954). The nominal
signicant level used was 0.05.

3. Results

1809

amount of food consumed in mg/kg body weight/day

were 12.4  3.04 (range 18.08.61), 125 30.0 (range
17686.4) and 1290  283 (1790917) for the 0.01, 0.10
and 1.00% dietary propyl paraben groups, respectively.
The nal body and reproductive organ weights are summarized in Table 1. There were no treatment-related
eects of propyl paraben on the testes, epididymides,
ventral prostates, seminal vesicles and preputial glands
(either absolute or relative to body weight) in any of the
groups.
3.2. Sperm reserves and sperm concentrations in cauda
epididymides and daily sperm production and eciency
of sperm production in testes
The cauda epididymal sperm reserves (number of
sperms/cauda epididymis) and sperm concentrations
(number of sperms/g cauda epididymis) decreased in a
dose-dependent manner and the dierence was signicant at a dose of 0.10% and above (Table 2). The
daily sperm production and its eciency in the testis
also decreased dose-dependently and the decrease was
signicant in all treated groups (Table 3).

3.1. Body and organ weights

3.3. Serum testosterone concentration
The amounts of food consumed were similar in the
four groups (Fig. 1). In the group with the highest
intake of propyl paraben, the body weight was slightly
but signicantly lower than that of the control group.
The average propyl paraben intakes calculated from the

The serum testosterone concentration decreased in a

dose-dependent manner (Table 4). The decrease was
signicant at a dose of 1.00% and the testosterone concentration at this dose was 64.6% of the control value.

Fig. 1. Changes in food intake of rats fed propyl paraben contained diets for 4 weeks. Values are represent mean for eight animals and error bars are
not shown for clarity. There were no statistical dierences among all groups.

1810

S. Oishi / Food and Chemical Toxicology 40 (2002) 18071813

Table 1
Summary of nal body weight and organ weight valuesa
Concentration

Body weight

Absolute weight
0.00%
0.01%
0.10%
1.00%

2768.60
28013.9
2747.17
26112.5*

Relative weight
0.00%
0.01%
0.10%
1.00%

Testes

Epididymides

Ventral prostates

Seminal vesicles

Preputial glands

2.65 0.222
2.67 0.165
2.60 0.203
2.60 0.150

0.3950.034
0.4050.029
0.4160.044
0.4100.061

0.2660.047
0.3100.055
0.2880.069
0.2610.016

0.5000.097
0.5590.087
0.5050.141
0.4730.131

0.1960.074
0.2070.035
0.1710.046
0.1850.032

0.961 0.077
0.955 0.063
0.950 0.088
0.999 0.021

0.1430.009
0.1450.010
0.1520.017
0.1570.021

0.0940.015
0.1110.022
0.1050.026
0.1000.017

0.1810.032
0.2000.028
0.1840.050
0.1820.052

0.0710.025
0.0740.010
0.0620.017
0.0720.016

* Signicantly dierent from control value, P<0.05.

a
Values are represent meanS.D. for eight animals.

Table 2
Sperm counts in the cauda epidydimis of rats exposed to propylparabena
Concentration (%)

Reserves (107/cauda)

Concentration (107/g)

0.00
0.01
0.10
1.00

43.6 18.2
31.1 10.5
25.7 6.97*
22.5 11.9*

10843.6
70.818.7
63.117.7*
48.826.6*

* Signicantly dierent from control, P <0.05.

a
Values are the meanS.D. for eight rats.

Table 3
Sperm counts in the testis of rats exposed to propylparabena
Concentration (%)

DSP (106)

Eciency (106)

0.00
0.01
0.10
1.00

37.55.32
26.22.34*
27.09.07*
25.93.90*

30.0 5.80
20.6 1.76*
22.4 8.95*
21.4 3.91*

* Signicantly dierent from control, P <0.05.

a
Values are the meanS.D. for eight rats.

Table 4
Testosterone concentration in the serum of rats fed propylparaben
contained diets for 4 weeksa
Testosterone (ng/ml)
0.00%
0.01%
0.10%
1.00%
* Signicantly dierent from control, P <0.05.
a
Values are mean S.D.

9.082.12
8.202.29
7.173.20
5.862.82*

4. Discussion
Most commercially available diets for laboratory animals are formulated with constituents that contain
phytoestrogens derived from plant materials, for example genistein and daidzein from soybean or coumesterol
from alfalfa. These phytoestrogens are estrogenic to
laboratory animals, which inuences the outcome of
endocrine toxicity evaluation (Boettger-Tong et al.,
1998; Odum et al., 2001). In the present experiment, a
phytoestrogen-free AIN93G modied diet was used in
order to avoid the estrogenic eects of phytoestrogens.
In the preliminary data, the growth rate did not dier
between rats fed the AIN93G diet and those fed commercially available CE-2 diet, which is manufactured by
Clea Japan Inc., Tokyo and is commonly used in this
laboratory.
There were no treatment-related eects of propyl
paraben on the sex organ weights (both absolute and
relative to body weight). However, the body weight was
slightly but signicantly lower in the group given 1.00%
propyl paraben than that in the control. Growth inhibition is a sensitive indicator of estrogenic eects and
has been used as a measure of estrogen potency (Heywood and Wadsworth, 1980; Hart, 1990). Matthews et
al. (1956) reported that consumption of a diet containing 2% propyl paraben (average intake of propyl paraben was 0.91.2 g/kg body weight/day) did not alter the
body weight. The reason for the discrepancies in these
results is not clear, but these may be due to various
factors, such as dierent diets, dierences in strains and
ages of animals used, and dierences in experimental
periods. These results in rats indicate a marginal eect
of parabens on general toxicological parameters at a
diet level of about 10 g/kg diet weight (nearly 10001300
mg/kg body weight), and the decreased body weight
induced by propyl paraben may be a toxic eect of
parabens rather than an estrogenic eect. As we reported previously (Oishi, 2001), the weights of epididymides

S. Oishi / Food and Chemical Toxicology 40 (2002) 18071813

and ventral prostates of rats administered with butyl

paraben were lower than those of the control. On the
other hand, the body weight of butyl paraben-treated
rats was slightly but not signicantly lower than that of
the control. The dierence in eects on organ weights
between rats administered with propyl paraben and
those with butyl parabens may be due to the dierence
in the dietary phytoestrogen content and/or the duration of exposure to parabens.
The sperm reserves and concentrations in the cauda
epididymis decreased with the increase in the dose of
propyl paraben, and the decreases were signicant at
dose of 0.10% or higher. In addition, the daily sperm
production and its eciency in the testis signicantly
decreased at a dose of 0.01%. The testosterone concentration in the serum decreased dose-dependently and
the decrease was signicant at a dose of 1.00%. The
development of the male reproductive system and spermatogenesis are controlled by testosterone (Desjardins,
1985; Herbert et al., 1995). The present results and our
previous report (Oishi, 2001) show that the dietary
concentration of butyl and propyl parabens, which
inuenced the sperm counts in the testis and epididymis,
was lower than the concentration at which the testosterone concentration in serum is aected. Although the
possibility that the decrease in the testosterone concentration may result in the decrease in sperm number
cannot be ruled out, the decreases in sperm number
seems to be a direct toxic eect on the spermatogenesis
or estrogenic action of parabens. For example, it has
been reported that several parabens exhibited spermatocidal activity via impairment of the sperm membrane
function in in vitro experiments (Song et al., 1989,
1991). Propyl paraben was administered at the onset of
spermatogenesis in the present study. The maternal
exposure of butyl paraben had adverse eects on sperm
production in F1 male rats (Kang et al., 2002). In addition, the long-term administration of butyl paraben also
caused the reduction of sperm counts in the testis and
epididymis (Oishi, 2002). These results suggest that the
reduction in the number of sperms is considered to be a
direct toxic and/or hormonal eect which may continue
to adulthood, and the possibility of the delay of sperm
development by propyl paraben cannot be denied.
Several in vitro studies showed that parabens are
estrogenic compounds. These studies demonstrated that
butyl ester showed the greatest activity. The activities of
other compounds are in the order propyl> ethyl> methyl
ester, the latter being only marginally active (Routledge et
al., 1998; Blaier et al., 2000; Nishihara et al., 2000; Okubo et
al., 2001; Satoh et al., 2000). Based on the sperm counts in
the testis and epididymis and testosterone concentration
in serum, however, it was demonstrated that the dose of
propyl paraben at which its eect is exerted is the same
as that of butyl paraben in vivo.

1811

Our ndings show that addition of parabens to the diet

of post-weaning male rats results in the disruption of
their reproductive system. The exposure level at which
this disruption is observed is the same as the upper-limit
acceptable daily intake (10 mg/kg body weight/day) of
parabens in the European Community and Japan (SCF,
1996; Suzuki et al., 1999). Because of the importance of
these eects, more detailed studies of the eects of parabens on the male reproductive organs will be necessary.

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