Institut National de Recherche et de Sécurité (INRS),
Vandoeuvre, France (J.-P.P., J.-P.F., D.B., I.B., E.F., F.C., M.-C.G.);
and Laboratoire de Dermatologie et de Cosmétologie, Faculté
de Pharmacie, Chatenay-Malabry, France (J.-P.M., C.M.V.)
 |
Introduction |
Di-n-Butylphthalate
is widely used. In 1994, total DBP1 production in
Western Europe and Japan was about 49,000 (World Health Organization,
1997
) and 17,000 tons (J. Property Investment and Finance, 1995),
respectively. In 1987, the USA production was 114,000 tons (National
Toxicology Program, 1995). DBP is extensively used as a plasticizer for
nitrocellulose, polyvinyl acetate, and polyvinyl chloride. DBP is not
covalently bound to polymeric matrix and is able to migrate. It is also
used as a solvent for printing inks or resins, and as a textile
lubricating agent or in cosmetics as a perfume solvent and a fixative
(World Health Organization, 1997).
The acute toxicity of DBP is low. Reported LD50
values following oral administration range from 8 to 20 g/kg in rats
(Smith, 1953; Lehman, 1955; White et al., 1980; Brandt, 1985)
and 5 to 16 g/kg in mice (Yamada, 1974; Brandt, 1985; Woodward,
1988). In rabbits, the dermal LD50 is
greater than 4 g/kg (Lehman, 1955).
After oral administration, DBP induces atrophy and testicular lesions
in male rats (Cater et al., 1977; Oishi and Hiraga, 1980; Gangolli,
1982; Gray et al., 1982; Fukuoka et al., 1989; Srivastava et al.,
1990). DBP has also been associated with developmental toxic effects in
mice and rats following oral (Nikoronow et al., 1973; Shiota and
Nishimura, 1982; Saillenfait et al., 1998) or intraperitoneal
administration (Singh et al., 1972; Peters and Cook, 1973).
Embryolethal and teratogenic effects were also reported in rats (Ema et
al., 1993, 1994, 1995b). It has been suggested that its main
metabolite, mono-n-butylphthalate, may be responsible partly
for the developmental toxicity of DBP (Ema et al., 1995a, 1996).
After oral administration of [14C]DBP to rats,
90 to 96% of the administered dose is excreted in urine within 48 h (Williams and Blanchfield, 1975; Tanaka et al., 1978). Phthalic acid,
monobutylphthalate (MBP), monobutylphthalate glucuronide (MBP-Gluc),
and mono-(3-hydroxybutyl) phthalate have been identified as metabolites
of DBP in urine (Williams and Blanchfield, 1975; Tanaka et al., 1978).
Following a single oral or intravenous dose of DBP to rats and
hamsters, MBP-Gluc was a common major metabolite (Foster et al., 1982), and unchanged DBP was at a low level in urine.
Assuming that oral exposure is prevented by personal hygienic measures,
the risk characterization for workers is limited to the dermal and
respiratory exposure paths. From in vivo study with rats, it has been
determined that approximately 10% of the applied dose of neat DBP is
absorbed per day, leading to a total absorption of ca. 72% within 7 days (Elsisi et al., 1989). The absorption flux was calculated to be 20 to 40 µg/cm2/h. From in vitro studies, the
absorption flux in rats determined in occluded or unoccluded conditions
was very similar (39-43 µg/cm2/h) (Mint and
Hotchkiss, 1993). A non-negligible amount of DBP remained within the
skin at the end of the experiment, which suggested that the skin might
be a reservoir for DBP. In human skin, the absorption flux was found to
be about 20- to 50-fold lower than that in rats (Scott et al., 1987;
Mint and Hotchkiss, 1993).
Although metabolism and excretion of DBP is well documented in animal
after oral administration, toxicokinetics and metabolism profiles have
received minimal investigations after either intravenous administration
or topical application. The current study was therefore carried out to
determine the toxicokinetic parameters and the metabolism of DBP after
intravenous and topical application to haired male rats. Additionally,
the percutaneous absorption between haired male and female rats or
hairless male rats was compared using in vivo and/or in vitro methods.
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Materials and Methods |
Chemicals.
Radiolabeled di-n-butyl
[carboxyl-14C]phthalate
([14C]DBP) was supplied by Amersham Pharmacia
Biotech UK, Ltd. (Buckinghamshire, England). It had a
radiochemical purity exceeding 97% and a specific activity of 26 mCi/mmol (960 MBq/mmol). Unlabeled DBP (99% pure) and MBP (99% pure)
were purchased from Merck (Darmstadt, Germany). All other reagents and
chemicals were obtained from commercial sources at the highest purity
available. Di-isopropylfluorophosphate (DIPFP) and Cremophor EL (a
derivative of castor oil and ethylene oxide) were from Sigma Aldrich
Chemie GmbH (Steinhem, Germany).
Animals.
Male or female haired and male hairless Sprague-Dawley rats (Iffa
Credo, Saint-Germain-sur-l'Arbresle, France) weighing 250 to 300 g were used for all studies. The animals were acclimatized to
laboratory conditions for at least 4 days prior to initiating the
studies in rooms with a 12-h light/dark cycle designed to control
relative humidity 50 ± 5%, and temperature 22 ± 1°C.
Commercial food pellet (UAR Alimentation-Villemoison, Epinay sur Orge,
France) and tap water were available ad libitum.
Intravenous Toxicokinetics of [14C]DBP in Haired
Male Rats.
Four days before the administration of the toxicant, a catheter was
introduced into the carotid artery of male rats. The tubing (PE 10, Biotrol, Paris, France) was passed s.c. exteriorized through the neck
and inserted into a protector stainless tubing (ca. 2 g weight)
that was ligatured firmly to the skin. Animals were placed into
individual plastic metabolic cages. Labeled
[14C]DBP (1 and 10 mg of DBP/kg) was
administered intravenously in a Cremophor suspension into the dorsal
vein of the penis of lightly etherized haired male rats
(n = 5-6). The individual doses were determined by
weighing the syringe before and after each administration. The
radioactivity concentrations were determined on 2 aliquots of each
solution. The radiocarbon dose was about 100 µCi/kg (3.7 MBq/kg).
After dosing, animals were immediately placed in individual metabolic
cages for collection of urine and feces at 4°C within 72 h. At
different times (10 s to 72 h), blood was collected from the
catheter into 20 µl of DIPFP, 10 mM (used as an inhibitor of
esterase) and 10 µl of heparin (5000 IU/ml, Choay, France). Blood was
immediately centrifuged (1 min; 12,000g). Plasma (ca. 150 µl) was removed quickly and introduced in tube containing 2 ml of 0.1 M acetate buffer, pH 1. A preliminary study showed that in these
conditions, the hydrolysis of spiked DBP in blood was minimal (<5% of
the spiked dose). At the end of the experiment, animals were sacrificed
by bleeding the abdominal aorta under light ether anesthesia.
Radioactivity content of the carcass was determined after digestion in
25% aqueous KOH solution (1:2, w/v).
Biliary Cannulated Rats.
Five days before a single intravenous administration (1 mg/kg in
Cremophor) or a topical application (10 µl/cm2,
10 cm2) of [14C]DBP, a
catheter was introduced into the carotid artery. Additionally, a
catheter was introduced into the bladder of rats dosed topically. This
catheter allowed the sequential collection of urine by administration of 2 ml of saline solution. The catheters were exteriorized at the back
of the neck, as described above. Just before the experiment, the common
bile duct was cannulated near the hilum of the liver under ether
anesthesia and the catheter was exteriorized back. Bile flow was
monitored for 1 h before administration of the toxicant. Blood,
bile, and urine were collected at different times within 30 h.
Rats had free access to food and water spiked with 0.9% w/v NaCl,
1.5% w/v glucose, and 0.05% KCl (Tse et al., 1982).
In Vivo Percutaneous Penetration and Absorption of
[14C]DBP.
One day before the dose was administered, the middle of the back of
haired male rats was clipped by an electric clipper, and a circular
ring (10 cm2) was glued. After topical
application of neat [14C]DBP (10 µl/cm2), the skin was covered by a perforated
circular plastic cap to allow aeration. Batches of three to eight
haired male rats were sacrificed at different times (0.5-72 h) after
the dosing by bleeding the abdominal aorta under light ether
anesthesia. Blood was collected on DIPFP and heparin. After sacrifice,
the skin area of the application site was washed five times with 200 µl of ethanol to remove the unabsorbed fraction of DBP (Mint and
Hotchkiss, 1993). A preliminary study showed that ethanol was more
efficient at removing unabsorbed DBP than soap solutions (95% of the
applied dose, n = 3). The radioactivity of the skin
area covered by the ring and that around the ring (about 30 cm2) was measured after digestion in KOH
solution. Radioactivity in carcass and excreta (urine and feces) was
also analyzed.
Sex and Strain Comparison of in Vivo Percutaneous Absorption of
DBP.
Percutaneous absorption was studied in haired male (n = 9), haired female (n = 5), and hairless male rats
(n = 6). A catheter was introduced into the bladder and
the carotid artery 4 days before, and the skin was clipped 1 day before
the topical application of neat [14C]DBP (10 µl/cm, 10 cm2). Twenty hours after dosing, the
unabsorbed dose of [14C] DBP was removed with
ethanol. Blood and urine were collected from the catheter at different
times as described above. Animals were sacrificed 72 h after the
[14C]DBP application. Radioactivity in plasma,
excreta, carcass, application skin area, and skin area around the ring
were determined by liquid scintillation.
In Vitro Percutaneous Absorption of DBP.
In vitro percutaneous absorption was assessed with static diffusion
cells using full-thickness skin from haired (n = 9) and hairless (n = 9) male rats. Rats were sacrificed with
pentobarbital. The whole dorsal region was shaved and the excess of
subcutaneous tissue carefully removed. The skin section was cut into
(1.76-cm2) circular sections (4 per rat) and
placed, stratum corneum side up, into diffusion cells. The diffusion
cells were maintained at a temperature of 36°C with a circulating
water bath, yielding a skin surface temperature of 32°C. The dermis
side was kept in contact with the receptor fluid (RPMI, Life
Technologies Europe, Paisley, Scotland) containing 2% albumin bovine
and 1% penicillin-streptomycin. The fluid receptor was previously
filtered through a sterile (Millex Millipore, Bedford, MA) 0.22-µm
pore size filter and degassed with a vacuum pump. Preliminary
experiments had shown that absorption flux was not significantly
different when the receptor fluid was NaCl 9%, NaCl 9%, and albumin
2%, or NaCl 9% and Volpo 20 4% as surfactant. The integrity of the
skin samples was assessed by determining the trans-epidermal
water lost (Tewameter, TM210, Courage + Khazaka) after an equilibrium
time of 1 h. An infinite dose of neat
[14C]DBP (50 mg/cm2) was
applied on a skin surface area of 1.76 cm2. An
aliquot (200 µl) of receptor fluid (5.15 ml) was collected periodically over a period of 24 h with an automatic fraction collector (Gilson FC 204, Middleton, WI). The cells were unoccluded. At
the end of the experiment, the unabsorbed dose of DBP was washed in
ethanol (1 × 500 µl). Radioactivity in receptor fluid, in
washing ethanol, and in exposed skin surface, after digestion in 25%
aqueous KOH solution (1:2, w/v), was measured. An additional
experiment was conducted with a sample of haired skin from three rats
and DIPFP 10 mM was added to the receptor fluid 1 h before
the dermal application of DBP.
HPLC Analysis of [14C]DBP Metabolites.
The method has been extensively described elsewhere (Saillenfait et
al., 1998). In short, an aliquot of plasma, bile, or urine in acetate
buffer was applied to a Sep Pak C18 cartridge
(Waters, Milford, MA). DBP, MBP, and its glucuronide conjugate were
eluted with 3.5 ml of a mixture of tetrahydrofuran and methanol (3:1, v/v). Ninety-seven percent of the plasma radioactivity from poisoned DBP rats was recovered in tetrahydrofuran-methanol mixture (97 ± 1.5%, n = 12). After partial concentration, the DBP
and its metabolites were analyzed by HPLC. The column was a reversed
phase Nucleosil C18 (SFCC-Shandon,
Neuilly-Plaisance, France). The elution was carried out with a
gradient of 0.01 M acetate solutions, pH 2.75, in acetonitrile. Eluate
radioactivity was measured on-line with a FlowOne spectrophotometer
(Packard, St. Louis, MO) or in 0.4-ml fraction (30 s). The HPLC
retention times of the radioactive peaks were compared with the
retention times of authentic standards (DBP, MPB) treated in the same
way. The retention time of the [14C]MBP
glucuronide was determined by comparing the chromatograph profiles of
plasma from poisoned [14C]DBP rats before and
after hydrolysis with a 
-glucuronidase from Escherichia
coli type IX (Sigma Chemical Co., St. Louis, MO). More than 99%
of the radioactivity applied to the HPLC column was recovered in the eluates.
Analysis of Radioactivity.
Samples of urine (1000 µl) and plasma (100-500 µl) were accurately
weighed and added directly to liquid scintillation vials containing 10 ml of liquid scintillation solution (Pico Fluor 30, Packard). Feces,
carcass, and skin sample were solubilized in a 25% aqueous solution of
KOH (1:2, w/v) for 3 days and homogenized. Aliquots of homogenates
(250-500 mg) were mixed with 10 ml of Pico Fluor 30. The radioactivity
of all the samples was measured in a Packard liquid scintillation
spectrophotometer model 1900. Efficiency of counting was determined by
quenching correction curves for the various addition and scintillation fluids.
Expression of Data and Statistical Analysis.
Values were expressed as mean ± S.E.M. The time course of
14C, DBP, MBP, and MBP-glucuronide concentrations
in plasma were fitted by a one- or two-exponential function with the
Kintool software (Qualilab, Orléans, France). The choice of the
model was based on the lower Akai index value. The elimination rate
constant in the central compartment (Kel)
and the terminal elimination rate () were obtained by log linear
concentration time data. The area under the plasma curves of DBP and
its metabolites from time 0 to the end of the experiment (AUC
0-t) were calculated by the linear trapezoidal
rule. The AUC from t to infinity was estimated by the calculated
concentration at t divided by . The sum of both areas was
AUC(0-inf). Clearance (CL) was the administered dose divided by the AUC(0-inf). The kinetics of
urinary 14C excretion were calculated with the
Sigma-minus method (Ritshel, 1980).
The percentage of the absorbed dose was calculated from
(a) the radioactivity content in excreta and carcass:
|
(1)
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(b) the ratio of the AUC of the total radioactivity, MBP, or
MBP-Gluc after topical application versus intravenous administration (1 mg/kg):
|
(2)
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(c) the ratio of the total radioactivity, MBP, or MBP-Gluc
excreted in urine after topical application versus intravenous administration (1 mg/kg)
|
(3)
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The percentage of the penetrated dose was calculated from
(a) the radioactivity content in excreta, carcass, and
skin:
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(4)
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Absorption flux (µg/cm2/h) was
calculated from
|
(5)
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|
(6)
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|
(7)
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Penetration flux (µg/cm2/h) was
calculated from
|
(8)
|
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Results |
Intravenous Administration.
Up to 72 h after a single i.v. administration of 1 or 10 mg/kg
[14C]DBP to haired male rats, the mean total
recovery of radioactivity in excreta and carcass amounted to 99.8% of
the dose (Table 1). Except a slight
higher level of radioactivity that remained in the carcass at the low
dose, no other significant differences were observed in the excretion
of 14C between the two doses. The main route of
excretion of 14C was in urine (85% of the
administered dose) followed by feces as a secondary route (9-11% of
the administered dose). More than 97% of the 14C
in urine was excreted in the first 24 h (Fig.
1). Unchanged DBP was barely detectable
in urine. Total urinary excretion of MBP did not significantly differ
between the two doses and accounted for 22.8 ± 3.7% of the
administered dose. Similar results were obtained for urinary excretion
of MBP-Gluc (34.6 ± 3.6% of the administered dose).
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TABLE 1
Mass balance of total radioactivity 72 h after a single
intravenous administration of [14C]DBP in haired male rats
Values are expressed as percentage of the administered dose (means ± S.E.M.).
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The time course in plasma of total 14C, unchanged
DBP, and its two main metabolites (MBP, MBP-Gluc) obtained after a
single i.v. administration of 1 and 10 mg/kg are shown in Fig.
2, A and B, respectively. The declines in
unchanged DBP and its two metabolites were best fitted by a
bi-exponential function. Initial levels of unchanged DBP in plasma,
determined from the equations of the plasma time curves, were 38 ± 4 and 770 ± 180 nmol/ml for the lowest and highest dose,
respectively. Unchanged DBP disappeared rapidly from the plasma. Thus,
30 min after the dose was administered, the concentration of DBP was
lower than the limit of quantitation (0.0015% of the dose, 0.015 nmol/ml for the 1-mg/kg dose). The half-life of unchanged DBP in the
terminal phase was about 5 to 7 min (Table
2). The fast elimination of DBP from the
plasma may result in part from esterase activity in the plasma. From in
vitro experiments, the half-life for hydrolysis of DBP in plasma was
calculated to be 1.9 ± 0.1 and 3.4 ± 0.0 min for initial
concentrations of 65 and 665 nmol/ml of
[14C]DBP, respectively (Fig.
3).
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TABLE 2
Plasma toxicokinetic parameters of 14C, unchanged DBP, and its
two main metabolites after a single intravenous administration of
[14C]DBP in haired male rats
The toxicokinetics parameters in plasma were determined by collection
of blood over a 72-h period after a single intravenous administration
of [14C]DBP (1 mg/kg or 10 mg/kg). Values are expressed as
means ± S.E.M.
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Peak concentrations of MBP and MBP-Gluc occurred between 1 to 2 and 20 to 30 min after the administration. The toxicokinetic parameters of
MBP-Gluc in plasma were not significantly different at the two doses of
DBP. In contrast, the maximal levels of unchanged DBP and MBP and their
respective AUC were proportionally higher at the 10-mg/kg dose than at
the 1-mg/kg dose.
In Vivo Percutaneous Penetration and Absorption of
[14C]DBP in Haired Male Rats.
In vivo percutaneous penetration and absorption of
[14C]DBP were determined by sacrificing the
animal at different times after a topical application of neat
[14C]DBP (10 µl/cm2).
DBP penetrated rapidly into the skin (Fig.
4). Thirty minutes after the topical
application, about 20% of the applied dose had entered the skin. This
percentage remained constant for 8 h and corresponded to 1.5 mg/cm2 of skin (Table
3). During this period, the absorbed dose
of DBP increased linearly with exposure time. The absorption flux calculated during this period was 43 µg/cm2/h
(Fig. 4). For exposure times of between 8 and 48 h, the absorption flux was 3.6 times higher (156 µg/cm2/h).
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TABLE 3
Mass balance of 14C after a topical application of
[14C]DBP in haired male rats
Batches of rats (n = 3-8) were sacrificed at different
times after a topical application of neat [14C]DBP (10 µl/cm2, 10 cm2). Values are expressed as means ± S.E.M.
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Whatever the exposure time, significant amounts of the applied
14C dose were in the skin around the application
site. For exposure times of 24 and 72 h, the concentrations of
14C in the skin at the application site and
around this area were not significantly different. This may be the
result of a radial diffusion of DBP into the corneum stratum and/or
epidermis. To test this hypothesis, a ring was glued to the back of the
rat (n = 3), and two areas of the skin around the ring
were desquamated. A large dose of DBP (100 µl/cm2) containing a colorant was deposed in
the center of the ring. A few hours after application, only intact skin
around the ring was colorized (result not shown).
14C radioactivity levels in plasma increased
progressively with exposure time for 24 h (Fig.
5). For exposure times of 24 and 48 h, the levels of 14C, MBP, and MBP-Gluc were not
significantly different. Whatever the exposure time, unchanged DBP
accounted for less than 2% of the plasma radioactivity and
corresponded to 0.23 nmol/ml after 24 h of exposure. MBP and
MBP-Gluc compounds accounted together for 61 to 85% of the plasma
radioactivity (mean: 70 ± 3%, n = 37). Except
for an exposure of 48 h, MBP levels in plasma were similar to or
higher than MBP-Gluc levels (mean ratio = 1.3 ± 0.05, n = 33). MBP and MBP-Gluc accounted together for 50 to
66% of the urinary radioactivity (mean 66 ± 1%,
n = 5 × 5) with no significant correlation with
time of exposure. The ratio of MBP to MBP-Gluc urinary excretion rates
was less than 1 (mean = 0.73 ± 0.08, n = 5 × 5) (Fig. 6). For a 72-h
exposure period, total urinary excretions of MBP and MBP-Gluc were
14.1 ± 1 and 20.0 ± 1.0% (n = 5) of the
applied dose, respectively (data not presented).
Urinary excretion rates of 14C, MBP, and MBP-Gluc
were not significantly different for the 8 to 24 and 24 to 48 h
collection periods (Fig. 6). The percutaneous absorption flux estimated
from urinary excretion rates of 14C, MBP, and
MBP-Gluc between 24 and 48 h of exposure rates was not
significantly different (mean = 103 ± 10 µg/cm2/h; Table
4). In contrast, the percutaneous
absorption flux determined from plasma levels at 24 h of
14C, unchanged DBP, MBP, and MBP-Gluc differed
greatly (23-161 µg/cm2/h).
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TABLE 4
Estimation of the percutaneous absorption flux of [14C]DBP
from plasma concentrations and urinary excretion rates of 14C,
unchanged DBP, and its two main metabolites in haired male rats
Values are expressed as mean ± S.E.M. (n = 5).
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Biliary Cannulated Rats.
After an intravenous administration of
[14C]DBP, plasma concentrations and biliary
excretion rates of total radioactivity declined similarly (Fig.
7). Within 30 h, about half the
administered dose was excreted either in urine or in bile (Table
5). More than 95% of the
14C in bile were excreted in the first 4 h.
After 8, 24, and 30 h of the dose being administered, MBP-Gluc
accounted for 81 ± 1% of the biliary radioactivity (data not
shown). Total urinary excretion of MBP and MBP-Gluc were 3.8 ± 2.3 and 24.6 ± 2.2% (n = 3) of the dose,
respectively.
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TABLE 5
Comparison of plasma content and excretion of total radioactivity in
bile- and non-bile duct-cannulated haired male rats after an
intravenous administration and a topical application of DBP
Values are expressed as means ± S.E.M.
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After a topical application of neat [14C] DBP,
plasma concentrations and biliary excretion rates of the total
radioactivity increased up to 8 h (Fig.
8) and remained roughly steady until 26 h of exposure. At the end of the experiment, the levels of MBP
and MBP-Gluc were not significantly different and together accounted
for 83 ± 1% of plasma radioactivity (data not presented). At
this time unchanged DBP represented little (0.2-3%) of the plasma
radioactivity. Whatever the time of exposure, the clearance of
14C in urine was higher than that in bile. Thus,
the mean of the ratio of the urinary excretion rates to the biliary
excretion rates was 2.9 ± 0.2 (n = 4 × 12).
For an exposure of 30 h, the total MBP and MBP-Gluc excreted in
urine were 4.1 ± 0.5 and 13.7 ± 0.7% of the applied dose
(n = 4), respectively.
Table 5 compares the results obtained in cannulated and non cannulated
bile duct rats dosed intravenously or topically. The AUC values of
total radioactivity concentration curves in plasma and
14C content in urine were significantly higher in
noncannulated rats than in cannulated bile duct rats dosed
intravenously. In contrast, the respective AUC values were very similar
in the two groups of rats dosed topically. Additionally, urinary
excretion was not as affected by bile duct cannulation in rats dosed
topically compared with those dosed intravenously. The percutaneous
flux estimated from the urinary and biliary excretion rate of
14C between 8 and 24 h was 131 ± 7 µg/cm2/h (n = 4).
In Vivo Sex and Strain Comparison of Percutaneous Absorption of
DBP.
The percutaneous absorption of DBP in haired male and female rats and
in hairless male rats was compared after exposure of 24 h to neat
[14C]DBP. The animals were sacrificed 48 h
after removing the unabsorbed fraction of DBP (Table
6). From the recovery of
14C in excreta and carcass, the percutaneous
absorption of DBP was not significantly different in haired male and
female rats and accounted for 56 to 61% of the applied dose. A large
fraction of the applied dose remained in the carcass and in the skin
48 h after washing the application site. Levels of
14C in plasma increased until the end of exposure
and decreased slowly thereafter (Fig. 9).
Particularly, the concentration of 14C in plasma
48 h after the end of exposure was about 44 and 31% of the peak
level for haired male and female rats, respectively. The peak of
14C concentration was higher in female rats than
in male rats. However, the apparent elimination rate of
14C in the plasma of male rats was slightly lower
than that in female rats. Thus, the AUCs extrapolated at an infinite
time were not significantly different between the two sexes.
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TABLE 6
Comparison of in vivo percutaneous absorption of [14C]DBP in
haired male and female or hairless male Sprague-Dawley rats
Twenty-four hours after a topical application of neat
[14C]DBP (10 µl/cm2, 10 cm2) the
application site skin was washed, and the animals were sacrificed
48 h later. Values are expressed as means ± S.E.M.
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The percutaneous absorption of DBP was higher in hairless male rats
(72% of the applied dose), with less than 5% of the applied dose
remaining in the carcass and skin (Table 6). 14C
concentration in plasma was not significantly different after 8 h
or 24 h of topical exposure. This result indicated that in steady-state conditions the percutaneous absorption occurred earlier than that in haired male rats. Moreover, after the end of exposure the
level of radioactivity in plasma decreased faster (3-fold) than that in
haired male rats. Similarly, the urinary excretion rate of
14C over the 8- to 24-h period was about 2.5-fold
higher in hairless male rats.
In haired rats, the absorbed dose estimated from the ratio of the
urinary excretion after a topical application to i.v. administration was not significantly different from the value obtained with the radioactivity contained in the carcass and eliminated in the excreta (Table 7). Similarly, the estimation of
the percutaneous absorption flux from the ratio of urinary excretion
rate between 8 and 24 h to the total urinary excretion after an
i.v. administration was not significantly different from the value
obtained by sacrificing the animals at different times after topical
exposure. However, as previously shown in Table 5, the percentage of
the absorbed dose and the percutaneous absorption flux determined from
the 14C plasma concentrations (AUC or
concentration at steady state) were largely underestimated (2-3-fold).
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TABLE 7
Estimation of the absorbed dose and percutaneous absorption flux of
[14C]DBP from urinary and plasma toxicokinetic parameters
Twenty-four hours after a topical application of neat
[14C]DBP (10 µl/cm2, 10 cm2) the
application skin site was washed, and the animals were sacrificed
48 h later. Values are expressed as means ± S.E.M.
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Similar results were obtained with hairless rats. Moreover, the
estimated percutaneous absorption flux appeared 2.5 times greater than
that of haired rats.
In Vitro Strain Comparison of Percutaneous Absorption of DBP.
In vitro percutaneous absorption experiments were conducted on haired
and hairless male rat skins with an infinite dose of [14C]DBP for 24 h. Percutaneous absorption
fluxes in haired and hairless male rats were 26 ± 1 and 39 ± 1 µg/cm2/h, respectively (Fig.
10). At the end of the experiment, all
the radioactivity contained in the receptor fluid from haired male rats
had the same HPLC retention time as the authentic MBP. Adding an
esterase inhibitor to the receptor fluid (DIPFP) caused a reduction of
99% of the absorption flux of 14C, and MBP was
barely detectable in the receptor fluid (result not presented).
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Discussion |
This report covers a study looking simultaneously at the plasma
toxicokinetics and excretion rates of total radioactivity, unchanged
DBP, and its two main metabolites (MBP and MBP-Gluc) after a single
intravenous administration and a topical application of
[14C]DBP in rats.
After an intravenous administration of [14C]DBP
(1 or 10 mg/kg), the parent compound was rapidly metabolized and
readily excreted in urine (85% of the administered dose) and to a
lesser extent in feces (ca. 10% of the administered dose). More or
less all the urinary excretion occurred within 24 h of the
administration being given. The two main urinary metabolites were MBP
and its glucuronide conjugate, together accounting for 57% of the
administered dose. These results are in accordance with previous
reports. In the rat, more than 90% of the dose was excreted in urine
within 48 h following intravenous administration of 10 mg/kg
(Tanaka et al., 1978) or an oral administration of 60 mg/kg (Tanaka et al., 1978) and 100 mg/kg (Williams and Blanchfield, 1975). In the
present study, however, a large part of a 1-mg/kg dose was first
excreted in bile as MBP-Gluc (ca. 80% of the biliary radioactivity). Thus, comparison of radioactivity in excreta or in plasma of bile duct-
and non-bile duct-cannulated rats showed that about 35% of the dose
underwent an enterohepatic recycling and was subsequently excreted in
urine. A very similar result was obtained after an oral administration
of DBP in rats (Tanaka et al., 1978), where about 50% of the dose was
excreted in bile. However, in this latter experiment, about half the
radioactivity eliminated in bile was unchanged DBP. Unchanged DBP was
also identified (but not quantified) in the bile of rats dosed orally
(Kaneshima et al., 1978). In contrast, in the present study, parent
compound DBP was not detected in bile after an i.v. dose of 1 mg of
DBP/kg (limit of detection 0.005% dose/ml). The main biliary
metabolite was MBP-Gluc (ca. 80% of biliary radioactivity). This
discrepancy may be the result of differences in the dosage levels used
and/or the method of administration. Thus, after an intravenous
administration of 500 mg/kg of DBP, only 10% of the dose was excreted
in bile within 5 h (Kaneshima et al., 1978). This excretion rate
of total radioactivity was 4-fold lower than the excretion rate
obtained in the present study with a 1-mg/kg dose, suggesting a
hepatobiliary saturation occurring at the higher dose. Hepatobiliary
saturation may explain the clearance of unchanged DBP and MBP in plasma
being about 2-fold lower at the 10-mg/kg dose than at the 1-mg/kg dose.
Similarly, total radioactivity was cleared from the body more quickly
after an oral administration of 0.27 g/kg than after a 2.31-g/kg dose (Williams and Blanchfield, 1975). These authors have also shown that
the distribution of total radioactivity is general through the body. In
the present study, the total volume of distribution of unchanged DBP
administered intravenously (1 mg/kg) represented about 82% of the
total body weight. This finding indicated that biodistribution of DBP
itself was extensive.
The disappearance of unchanged DBP was very fast. Thus, half an hour
after administration, the level of unchanged DBP was barely detectable
in plasma. As DBP was not detected in urine or bile, the disappearance
of unchanged DBP from the plasma was the consequence of its
distribution throughout the body and its metabolism. A part of the
metabolism of DBP is likely to occur in the plasma. Thus, the half-life
of the elimination phase of unchanged DBP (5-7 min) was half of its
hydrolysis rate determined in vitro. Plasma hydrolysis of DBP might
explain the fast increase of MBP in plasma with a peak level, which
occurred as little as 1 to 2 min after the administration of the
toxicant. In contrast, its glucuronide conjugate, which was formed in
part in the liver, had a peak level that occurred later (20-30 min
after the administration).
After a topical application of neat DBP, the compound penetrated
rapidly into the skin. Thirty minutes after the topical application, about 20% of the applied dose had entered the skin. This percentage remained constant for 8 h and corresponded to 1 to 2 mg of
DBP/cm2. During this period of exposure, the
absorbed dose of 14C increased linearly with
time. The absorption flux calculated during this period was 43 µg/cm2/h. This value agrees closely with the
flux determined in vitro and in vivo in male Fischer 344 rat. The flux
determined in vitro with full thickness skin was 39 to 43 µg/cm2/h (Mint and Hotchkiss, 1993). From the
in vivo results reported by Elsisi et al. (1989), the flux was
calculated to be about 30 µg/cm2/h (dose = 157 µmol/kg; body weight = 0.2 kg; skin area = 1.33 cm2; urinary excretion rate per 24 h = 10-12%; urinary fraction = 0.85 from the present study).
However, for exposure times of 8 to 48 h, the absorption flux was
3.6 times higher (156 µg/cm2/h). This increase
in flux with exposure time was assumed to be due to radial diffusion of
the compound through the stratum corneum and/or the epidermis, leading
to an increase in the area of skin exposed. The hypothesis of radial
diffusion for DBP was based on the progressive increase in the
radioactivity content in the skin around the ring with exposure time.
For 24 h or 48 h of exposure, the radioactivity contents in
the area of deposit and around this area were very similar.
Additionally, when the skin was desquamated, the compound did not
diffuse outside the area of deposit.
For 48 h of exposure, 14C, DBP, MBP, and
MBP-Gluc plasma levels and the urinary excretion rate were not
significantly different from or just slightly lower than that for a
24-h exposure period. These results indicate that the percutaneous
absorption and the excretion mechanisms were in equilibrium over these
exposure periods. Thus, the percutaneous absorption rates in steady
state can be estimated from plasma concentration levels or urinary
excretion rates of DBP and its main metabolite after 24 h of
exposure. The percutaneous absorption flux estimated from the urinary
excretion rate of DBP and its metabolites gave quite similar values
(about 105 µg/cm2/h). This value was lower than
the value determined from radioactivity contents in excreta and carcass
(156 µg/cm2/h). This difference might be the
result of an underestimation of the percutaneous absorption rate
determined from radioactivity in excreta and in carcass. Thus, the
radioactivity content in carcass for a 24- or 48-h exposure period was
significant, and it can not be ruled out that a part of the
radioactivity content measured in carcass was not absorbed into the
systemic circulation but was in the skin.
The percutaneous absorption fluxes, estimated from plasma levels of
14C, DBP, and its metabolites after 24 h of
exposure, were quite different. In particular, percutaneous penetration
fluxes, estimated from unchanged DBP and total radioactivity, were 10- and 2.5-fold lower than that of the flux determined from radioactivity
in excreta and carcass, respectively. These findings suggest an
intensive skin first pass effect. As unchanged DBP in plasma was below
the limit of quantification, the determination of absolute
bioavailability from the ratio of the AUC of unchanged DBP after dermal
to i.v. exposure was imprecise.
Also, the hypothesis of skin first pass effect was verified
qualitatively and quantitatively in vivo and in vitro, respectively. As
previously discussed, after an i.v. administration about 35% of the
administered dose undergoes enterohepatic recycling before being
excreted in urine. In contrast, after dermal application, enterohepatic
recycling appears minimal. The fractions of the administered dose
excreted in urine and the AUC of total radioactivity in plasma were
very similar in cannulated and noncannulated bile duct rats. This
finding indicates that biodistribution of a part of the radioactivity,
which was available in the systemic circulation, did not have the same
biodistribution as unchanged DBP. Experiments conducted in vitro agree
closely with an extensive metabolism of DBP within the skin. Actually,
all the radioactivity contained in the receptor fluid was MBP.
Moreover, adding DIPFP, an inhibitor of esterase, drastically reduced
the appearance of radioactivity in the receptor fluid. Similarly,
intensive skin metabolism has been reported for butylbenzoic ester. In
contrast, percutaneous penetration of propylbenzoic ester was less
influenced by inhibition of skin esterase activity (Bando et al.,
1997).
A high first past effect has been reported after oral administration.
Based on in vitro studies, it is estimated that only 5% of the orally
absorbed dose is unchanged DBP. The main part is absorbed like the
monoester after enzymatic hydrolysis in the small intestines (White et
al., 1980). Additionally, whatever the time after an oral
administration of DBP to pregnant rats, unchanged DBP levels in
maternal plasma were barely detectable and accounted for less than 1%
of the radiocarbon activity (Saillenfait et al., 1998).
Percutaneous penetration was compared between male and female haired
rats and hairless male rats sacrificed 48 h after 24 h of
exposure. Differences between the two sexes were minimal. After
washing, the plasma radioactivity decreased slowly and the urinary
excretion rates determined 24 or 48 h after washing were about the
same. These results confirm the hypothesis that skin is a reservoir for
lipophilic phthalates (Mint and Hotchkiss, 1993). In vivo and in vitro
experiments have shown that the absorption flux is higher in hairless
male rats than in haired male rats. The higher permeability of rat skin
compared with human skin was attributed in general to a higher density
of appendage or lower thickness of rat skin versus human skin (Kao et
al., 1988; Illel et al., 1991). These two parameters cannot explain the
higher penetration flux of DBP in hairless male rats. The number of
hair follicles of hairless rats was lower, whereas the skin thickness of hairless and haired rats was not significantly different.
Similarly, it is accepted that percutaneous penetration increases with
increasing lipophilicity. Thus, in vivo percutaneous absorption of
diethylphthalate (log P octanol/water = 2.5) was lower than DBP
(log P octanol/water = 4.9) in rats (Elsisi et al., 1989).
However, a contradictory result was obtained in vitro with rat or human
epidermis (Scott et al., 1987) and in human skin (Mint et al., 1994).
As physicochemical or physiological factors can not explain the
interspecies and/or intercompound differences of the penetration flux
of phthalates, further studies are being conducted to determine the
influence of skin metabolism activity on the percutaneous rate of
different phthalates.
In conclusion, in vivo and in vitro results were very similar. They
have shown that DBP penetrated rapidly and diffused into the stratum
corneum and/or epidermis, which constituted a reservoir. From this
reservoir, DBP was slowly hydrolyzed by skin esterase before it reached
the systemic circulation. Skin reservoir, high lag time, and lower
excretion rate should be taken into account for risk assessment purposes.
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Abstract
Introduction
), MBP (
), and
MBP-Gluc (×) after a single i.v. administration of 1 mg/kg DBP (---)
or 10 mg/kg DBP (
). Unchanged DBP levels were lower than the limit of
quantification (0.0002% dose/ml).
, unchanged DBP; 
) or 64 nmol/ml of
[14C]DBP (
0.20 (±0.00)1 × t]; t1/2 = 3.4 ± 0.0 min. S2 (nmol/ml) = 65 (±1) × exp[
, haired male
(n = 9); ×, haired female (n = 4); 
, haired
male rats;