0090-9556/01/2906-843–854$3.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:843–854, 2001
Vol. 29, No. 6
263/905020
Printed in U.S.A.
IN VIVO AND IN VITRO PERCUTANEOUS ABSORPTION OF [14C]DI-N-BUTYLPHTHALATE
IN RAT
JEAN-PAUL PAYAN, JEAN-PAUL MARTY, JEAN-PAUL FABRY, DOMINIQUE BEYDON, ISABELLE BOUDRY,
ELISABETH FERRARI, FRANCINE CANEL, MARIE-CHRISTINE GRANDCLAUDE, AND CLAIRE MARIE VINCENT
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.)
(Received October 16, 2000; accepted February 8, 2001)
This paper is available online at http://dmd.aspetjournals.org
ABSTRACT:
(mainly as MBP-Gluc) and underwent hepatobiliary recycling. Unchanged DBP was barely detectable in excreta. DBP rapidly penetrated the skin, which constituted a reservoir. The absorption flux
determined for 0.5 to 8 and 8 to 48 h of exposure were 43 and 156
␮g/cm2/h, respectively. The higher flux may be due to radial diffusion of DBP in the stratum and/or epidermis. The in vivo and in vitro
experiments revealed that DBP was intensively metabolized into
the skin. In vivo percutaneous absorption flux was very similar in
male and female haired rats. In contrast, the percutaneous absorption determined in vivo and in vitro was higher in hairless than in
haired male rats. Absorption flux was accurately estimated from
urinary excretion rate of MBP or MBP-Gluc.
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%
1
Abbreviations used are: DBP, di-n-butylphthalate; [14C]DBP, [14C]di-n-butylphthalate; MBP, monobutylphthalate; MBP-Gluc, glucuronide conjugate of
MBP; DIPFP, di-isopropylfluorophosphate; HPLC, high-performance liquid chromatography; AUC, area under curve; AUC0–inf (i.v.), area under the plasma time
curve to infinity, slope of the terminal phase; CL, clearance; Cp, extrapolated
plasma concentration at time T ⫽ 0; Kel, total elimination rate constant in the
central compartment; MRT, mean residence time; % Qo, percentage of the
administered dose; Tmax, time for maximal plasma concentration; Vc, volume of
the central compartment.
Send reprint requests to: Dr. Jean-Paul Payan, Institut National de Recherche et de Sécurité, Avenue de Bourgogne, B.P. N° 27, 54501 Vandoeuvre Cedex,
France. E-mail: [email protected]
843
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This study evaluated the toxicokinetics of [14C]di-n-butylphthalate
([14C]DBP) after an intravenous administration (1 and 10 mg/kg, in
Cremophor) or a topical application (10 ␮l/cm2; 10 cm2, neat) in
haired male Sprague-Dawley rats. Additional in vivo and in vitro
percutaneous penetration studies of [14C]DBP were conducted on
male and female haired rats and male hairless rats. After intravenous administration, unchanged DBP disappeared rapidly from the
plasma, following a two-exponential function (T1/2␤ ⴝ 5–7 min).
The peak levels of monobutylphthalate (MBP) and its glucuronide
conjugate (MBP-Gluc) occurred 1 to 2 and 20 to 30 min after
administration, respectively. These metabolites were intensively
and rapidly excreted in urine (57% of the dose). However, about
35% of the dose recovered in urine was primarily excreted in bile
844
PAYAN ET AL.
within 7 days (Elsisi et al., 1989). The absorption flux was calculated
to be 20 to 40 ␮g/cm2/h. From in vitro studies, the absorption flux in
rats determined in occluded or unoccluded conditions was very similar (39 – 43 ␮g/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.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
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 ␮Ci/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 ␮l of DIPFP, 10
mM (used as an inhibitor of esterase) and 10 ␮l of heparin (5000 IU/ml,
Choay, France). Blood was immediately centrifuged (1 min; 12,000g). Plasma
(ca. 150 ␮l) 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 ␮l/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 ␮l/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 ␮l 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 ␮l/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% penicillinstreptomycin. The fluid receptor was previously filtered through a sterile
(Millex Millipore, Bedford, MA) 0.22-␮m 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 ␮l) 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 ␮l).
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). Ninetyseven 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-Plai-
845
TOXICOKINETICS OF DBP
(a) the radioactivity content in excreta and carcass:
% of the absorbed dose
⫽ % of the applied dose in urine ⫹ feces ⫹ carcass
(1)
(b) the ratio of the AUC of the total radioactivity, MBP, or MBP-Gluc after
topical application versus intravenous administration (1 mg/kg):
% of the absorbed dose
⫽
AUC0 –inf topical application 共% dose/ml ⫻ h兲 ⫻ 100
AUC0 –inf(iv)共% dose/ml ⫻ h兲
(2)
(c) the ratio of the total radioactivity, MBP, or MBP-Gluc excreted in urine
after topical application versus intravenous administration (1 mg/kg)
% of the absorbed dose ⫽
% of the topical dose in urine ⫻ 100
% of the intravenous dose in urine
(3)
The percentage of the penetrated dose was calculated from
Absorption flux
⫽
% of the applied dose/ml in plasma at steady state ⫻ applied dose 共␮g/cm2兲
AUC0 –inf(iv)
(6)
urinary excretion rate at steady state
共% of applied dose/h兲 ⫻ applied dose 共␮g/cm2兲
Absorption flux ⫽
fraction of intravenous dose excreted in urine ⫻ 100
(7)
Penetration flux (␮g/cm /h) was calculated from
2
Penetration flux ⫽
% of the penetrated dose ⫻ applied dose 共␮g/cm2兲
time of exposure ⫻ 100
(8)
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).
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).
Peak concentrations of MBP and MBP-Gluc occurred between 1 to
TABLE 1
(a) the radioactivity content in excreta, carcass, and skin:
Mass balance of total radioactivity 72 h after a single intravenous administration
of [14C]DBP in haired male rats
% of the penetrated dose ⫽ % of the applied dose in
Values are expressed as percentage of the administered dose (means ⫾ S.E.M.).
urine ⫹ feces ⫹ carcass ⫹ application site skin ⫹ skin area around the ring
Excretion
1 mg/kg
(n ⫽ 5)
10 mg/kg
(n ⫽ 6)
Urine
Feces
Carcass
Cage washing
Collected blood
Recovery
85.2 ⫾ 0.9
9.3 ⫾ 1.0
2.0 ⫾ 0.1
0.4 ⫾ 0.0
1.9 ⫾ 0.2
98.7 ⫾ 0.6
85.8 ⫾ 2.4
10.8 ⫾ 1.1
0.6 ⫾ 0.1*
1.1 ⫾ 0.4
2.3 ⫾ 0.2
100.7 ⫾ 1.7
(4)
Absorption flux (␮g/cm2/h) was calculated from
Absorption flux ⫽
% of the absorbed dose ⫻ applied dose 共␮g/cm2兲
time of exposure ⫻ 100
(5)
* Significant difference from the 1-mg/kg dose ( p ⬍ 0.05).
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sance, 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 ␮l) and plasma (100 –
500 ␮l) 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
846
PAYAN ET AL.
Values are expressed as mean ⫾ S.E.M. (n ⫽ 5– 6). Urinary excretion of [14C]
(䡺), MBP (E), 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).
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 ␮l/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 ␮g/cm2/h (Fig. 4). For exposure
times of between 8 and 48 h, the absorption flux was 3.6 times higher
(156 ␮g/cm2/h).
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 ␮l/cm2) containing a colorant was
FIG. 2. Plasma time-curve of [14C]DBP after a single intravenous administration
in haired male rats.
Values are expressed as means ⫾ S.E.M. Symbols without bars indicate that
S.E.M. is within the symbol. A, 1 mg/kg (n ⫽ 5); B, 10 mg/kg (n ⫽ 3). 䡺, 14C; ‚,
unchanged DBP; E MBP; ⫻, MBP-Gluc. L.O.Q., limit of quantification (0.0015%
of dose/ml).
deposed in the center of the ring. A few hours after application, only
intact skin around the ring was colorized (result not shown).
14
C 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
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FIG. 1. Cumulative urinary excretion of unchanged DBP and its main
metabolites after a single i.v. administration of [14C]DBP in haired male rats.
847
TOXICOKINETICS OF DBP
TABLE 2
Plasma toxicokinetic parameters of
14
C, 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.
14
C
(n ⫽ 5)
3.6 ⫾ 0.4
3.4 ⫾ 0.6
0.039 ⫾ 0.001
1.83 ⫾ 0.15
1.77 ⫾ 0.06
1.82 ⫾ 0.06
96.9 ⫾ 9
55.2 ⫾ 1.9
12.7 ⫾ 0.7
(n ⫽ 6)
9.4 ⫾ 2.2*
7.39 ⫾ 0.92
132 ⫾ 22
0.024 ⫾ 0.003
0.026 ⫾ 0.003
132 ⫾ 46
4100 ⫾ 435
0.06 ⫾ 0.01
(n ⫽ 3)
10.3 ⫾ 4.6*
0.021 ⫾ 0.001
5.9 ⫾ 1.3*
1.46 ⫾ 0.1*
1.52 ⫾ 0.1*
59 ⫾ 16*
17.7 ⫾ 2.1*
65 ⫾ 5
5.96 ⫾ 0.48
222 ⫾ 70
0.045 ⫾ 0.009*
0.046 ⫾ 0.009*
73 ⫾ 40*
0.05 ⫾ 0.02
2347 ⫾ 440*
MBP
(n ⫽ 5)
MBP-Gluc
(n ⫽ 5)
0.03 ⫾ 0.003
0.50 ⫾ 0.03
0.20 ⫾ 0.05
2.1 ⫾ 0.2
0.26 ⫾ 0.02
0.28 ⫾ 0.03
0.33 ⫾ 0.04**
0.18 ⫾ 0.02**
0.15 ⫾ 0.05
0.77 ⫾ 0.12**
0.31 ⫾ 0.03**
0.40 ⫾ 0.09**
2.40 ⫾ 0.7
(n ⫽ 3)
6.8 ⫾ 2.7**
(n ⫽ 3)
0.023 ⫾ 0.006
0.90 ⫾ 0.06*
0.20 ⫾ 0.002
1.64 ⫾ 0.4
0.59 ⫾ 0.04*
0.60 ⫾ 0.04*
0.46 ⫾ 0.03**
0.18 ⫾ 0.02**
0.14 ⫾ 0.034
0.82 ⫾ 0.15**
0.36 ⫾ 0.03**
0.40 ⫾ 0.04**
1.43 ⫾ 0.06
5.29 ⫾ 0.5**
% Q0, percentage of the administered dose (1% Q0 ⫽ 10.7 nmol for the 1-mg/kg dose).
* Significant difference from the 1-mg/kg dose ( p ⬍ 0.05).
** Significant difference from MBP values at the same dose of [14C]DBP ( p ⬍ 0.05).
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
␮g/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 ␮g/cm2/h).
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.
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 ␮g/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.
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 radioac-
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
DOSE 1 mg/kg
Cp(t⫽0) (% Q0/ml)
tmax (h)
Cpmax (% Q0/ml)
␤ (1/h)
Kel (1/h)
AUC0–72 h(i.v.) (% Q0/ml ⫻ h)
AUC0–inf(i.v.) (% Q0/ml ⫻ h)
Vc (ml/kg weight)
CL (ml/h)
MRT (h)
DOSE 10 mg/kg
Cp(t⫽0) (% Q0/ml)
tmax (h)
Cpmax (% Q0/ml)
␤ (1/h)
Kel (1/h)
AUC0–72 h(i.v.) (% Q0/ml ⫻ h)
AUC0–inf(i.v.) (% Q0/ml ⫻ h)
Vc (ml/kg weight)
MRT (h)
CL (ml/h)
Unchanged DBP
(n ⫽ 5)
848
PAYAN ET AL.
Values are expressed as mean ⫾ S.E.M. (n ⫽ 3– 8). Batches of animals were
sacrificed at different times after a topical dose of neat [14C]DBP (10 ␮l/cm2; 10
cm2) cumulative penetration (f), and absorption (E). F1 (␮g/cm2/h) ⫽ 43 ⫾ 2; tlag1
(h) ⫽ 0. F2 (␮g/cm2/h) ⫽ 156 ⫾ 11; tlag2 (h) ⫽ 4.8.
FIG. 3. In vitro hydrolysis of DBP in plasma of haired male rats.
Values are expressed as mean ⫾ S.E.M. (n ⫽ 3). Plasma of rats was spiked with
664 nmol/ml (f) or 64 nmol/ml of [14C]DBP (䡺). An aliquot of plasma was
sampled at different times, and DBP concentration was determined by HPLC. S1
(nmol/ml) ⫽ 665 (⫾1) ⫻ exp[⫺0.20 (⫾0.00)1 ⫻ t]; t1/2 ⫽ 3.4 ⫾ 0.0 min. S2
(nmol/ml) ⫽ 65 (⫾1) ⫻ exp[⫺0.36 (⫾0.02) ⫻ t]; t1/2 ⫽ 1.9 ⫾ 0.1 min.
tivity 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).
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 ␮g/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).
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-cannu-
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 4. In vivo percutaneous cumulative penetration and absorption of neat
[14C]DBP in haired male rats.
14
TABLE 3
C after a topical application of [14C]DBP in haired male rats
0.2 ⫾ 0.0
0.1 ⫾ 0.0
18.4 ⫾ 0.3
4.6 ⫾ 1.2
74.5 ⫾ 2.2
97.8 ⫾ 0.6
0.3 ⫾ 0.3
0.06 ⫾ 0.01
23.3 ⫾ 1.6
4.8 ⫾ 0.6
0.0005 ⫾ 0.0001
1.84 ⫾ 0.03
0.15 ⫾ 0.04
0.3 ⫾ 0.1
0.2 ⫾ 0.1
15.5 ⫾ 2.3
3.0 ⫾ 0.8
79.4 ⫾ 2.8
98.8 ⫾ 0.0
0.5 ⫾ 0.1
0.06 ⫾ 0.01
19.4 ⫾ 2.8
2.0 ⫾ 0.3
0.0013 ⫾ 0.0001
1.58 ⫾ 0.23
0.13 ⫾ 0.03
1h
(n ⫽ 3)
c
b
2h
(n ⫽ 3)
1.0 ⫾ 0.2
0.1 ⫾ 0.0
11.2 ⫾ 1.4
6.8 ⫾ 3.5
79.2 ⫾ 3.2
98.4 ⫾ 1.0
1.1 ⫾ 0.1
0.06 ⫾ 0.01
19.1 ⫾ 2.3
1.0 ⫾ 0.1
0.0017 ⫾ 0.0001
1.12 ⫾ 0.14
0.21 ⫾ 0.11
Percentage of the applied dose.
Absorption flux (mg/cm2/h) ⫽ % of the absorbed dose/100 ⫻ dose (10.5 mg/cm2)/time of exposure.
Penetration flux (mg/cm2/h) ⫽ % of the penetrated dose/100 ⫻ dose (10.5 mg/cm2)/time of exposure.
d
Percentage of the applied dose per ml of plasma.
e
Percentage of the applied dose per cm2 of skin.
a
Urine
Fecesa
Carcassa
Cage washinga
Application site skina
Skin around the application sitea
Washing of the application site skina
Recoverya
Percentage of absorbed dosea
Absorption fluxb
Percentage of penetrated dosea
Penetration fluxc
Plasmad
Application site skine
Skin around the application sitee
0.5 h
(n ⫽ 3)
FIG. 5. Time course of total 14C, unchanged DBP, and its two main metabolites
in plasma after a topical application of neat [14C]DBP to haired male rats.
Values are expressed as mean ⫾ S.E.M. (n ⫽ 3– 8). f, 14C; ‚, unchanged DBP;
E, MBP; ⫻, MBP-Gluc. 1 nmol-Eq ⫽ 0.0003% of the applied dose. L.O.Q., limit
of quantification (0.15 nmol/ml). L.O.D., limit of detection (0.22 nmol/ml).
lated 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
8h
(n ⫽ 8)
0.6 ⫾ 0.2
0.0 ⫾ 0.0
2.2 ⫾ 0.5
0.4 ⫾ 0.1
14.4 ⫾ 2.9
1.7 ⫾ 0.3
79.9 ⫾ 2.6
99.4 ⫾ 0.2
3.3 ⫾ 0.5
0.04 ⫾ 0.01
19.4 ⫾ 2.4
0.3 ⫾ 0.0
0.0039 ⫾ 0.0004
1.44 ⫾ 0.29
0.06 ⫾ 0.01
0.4 ⫾ 0.0
0.0 ⫾ 0.0
1.3 ⫾ 0.2
0.3 ⫾ 0.0
17.0 ⫾ 2.8
2.5 ⫾ 0.5
79.3 ⫾ 3.5
100.8 ⫾ 2.0
1.9 ⫾ 0.2
0.04 ⫾ 0.01
21.5 ⫾ 2.9
0.5 ⫾ 0.1
0.0027 ⫾ 0.0002
1.70 ⫾ 0.28
0.09 ⫾ 0.01
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
4h
(n ⫽ 8)
Application Time
Batches of rats (n ⫽ 3– 8) were sacrificed at different times after a topical application of neat [14C]DBP (10 ␮l/cm2, 10 cm2). Values are expressed as means ⫾ S.E.M.
Mass balance of
12.1 ⫾ 1.2
1.0 ⫾ 0.1
14.7 ⫾ 2.3
3.25 ⫾ 0.7
4.2 ⫾ 0.4
12.9 ⫾ 1.0
46.6 ⫾ 5.3
94.7 ⫾ 1.0
31.0 ⫾ 4.3
0.13 ⫾ 0.0
48.1 ⫾ 4.6
0.2 ⫾ 0.0
0.0109 ⫾ 0.0012
0.43 ⫾ 0.03
0.46 ⫾ 0.04
24 h
(n ⫽ 8)
39.9 ⫾ 5.5
4.5 ⫾ 0.3
12.8 ⫾ 0.9
6.2 ⫾ 1.0
2.2 ⫾ 0.2
9.2 ⫾ 1.3
17.9 ⫾ 1.2
92.6 ⫾ 1.3
63.3 ⫾ 3.1
0.14 ⫾ 0.01
74.7 ⫾ 2.2
0.16 ⫾ 0.00
0.0083 ⫾ 0.000
0.22 ⫾ 0.06
0.40 ⫾ 0.03
48 h
(n ⫽ 4)
52.8 ⫾ 2.6
7.6 ⫾ 0.6
7.7 ⫾ 0.6
5.5 ⫾ 0.8
2.6 ⫾ 0.6
4.9 ⫾ 0.9
13.3 ⫾ 1.8
94.5 ⫾ 0.4
73.6 ⫾ 2.9
0.11 ⫾ 0.00
81.2 ⫾ 1.8
0.12 ⫾ 0.00
0.005 ⫾ 0.000
0.26 ⫾ 0.06
0.21 ⫾ 0.03
72 h
(n ⫽ 8)
TOXICOKINETICS OF DBP
849
850
PAYAN ET AL.
Values are expressed as mean ⫾ S.E.M. (n ⫽ 5). f, 14C; E, MBP; ⫻, MBPGluc. 1 ␮mol-Eq ⫽ 0.3% of the applied dose. Unchanged DBP levels were lower
than the limit of detection (0.4 nmol/ml).
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 conse-
TABLE 4
Estimation of the percutaneous absorption flux of [14C]DBP from plasma concentrations and urinary excretion rates of
metabolites in haired male rats
14
C, unchanged DBP, and its two main
Values are expressed as mean ⫾ S.E.M. (n ⫽ 5).
14
Unchanged DBP
MBP
1.82 ⫾ 0.06
85.2 ⫾ 2.9
0.026 ⫾ 0.003
0.28 ⫾ 0.03
25.7 ⫾ 2.5
0.011 ⫾ 0.002
0.88 ⫾ 0.05
0.00006 ⫾ 0.0000
0.0045 ⫾ 0.0007
0.24 ⫾ 0.02
60 ⫾ 12
23 ⫾ 3*
161 ⫾ 37*
113 ⫾ 34*
93 ⫾ 12
113 ⫾ 12
C
i.v.
AUC0–inf(i.v.) in plasmaa
Urine
Percutaneous
Cp24h (% dose/ml)
du/dt24–48h (% dose/h)
From plasma results
Absorption flux (␮g/cm2/h)
From urinary results
Absorption flux (␮g/cm2/h)
103 ⫾ 7
MBP-Gluc
0.40 ⫾ 0.03
33.5 ⫾ 2.5
0.0045 ⫾ 0.0009
0.38 ⫾ 0.027 (n ⫽ 5)
Cp24h(percut), concentration in plasma of 14C, unchanged DBP, or its two main metabolites after 24 h of a topical application of neat [14C]DBP (10 ␮l/cm2, 10 cm2). % Q0, percentage of the
administered dose (i.v.) or the applied dose (percutaneous).
* Significant difference from the value determined with 14C plasma level ( p ⬍ 0.05).
a
AUC0 –inf(i.v.), area under the plasma time curve of 14C, unchanged DBP, or its two main metabolites after a single i.v. administration of [14C]DBP 1 mg/kg in haired male rats (Table 2).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 6. Urinary excretion rate of total 14C and the two main metabolites of DBP
after a topical application of neat [14C]DBP to haired male rats.
quence 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 ␮g/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 ␮g/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 ␮g/cm2/h (dose ⫽ 157
␮mol/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 ␮g/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 ␮g/cm2/h). This
851
TOXICOKINETICS OF DBP
FIG. 8. Time course of plasma concentration, urinary, and biliary excretion rates
of total radioactivity after a topical application of neat [14C]DBP in haired male
rats.
Bile duct-cannulated haired male rats were dosed intravenously with 1 mg of
[ C]DBP/kg. Blood and bile were collected at different times from a catheter
introduced into the carotid artery and bile duct, respectively. f, plasma concentration of total radioactivity; the biliary excretion rate (E) was calculated at the
midpoint of each collection period. Values are expressed as mean ⫾ S.E.M. (n ⫽ 3).
Bile duct-cannulated haired male rats were dosed topically with neat [14]DBP (10
␮l/cm2; 10 cm2). Blood, bile, and urine were collected at different times from
catheters introduced into the carotid artery, the bile duct, and the bladder, respectively. f, plasma concentration of total radioactivity; biliary (⫻) and urinary (E)
excretion fluxes were calculated at the midpoint of each collection period. Values
are expressed as mean ⫾ S.E.M. (n ⫽ 4).
value was lower than the value determined from radioactivity contents
in excreta and carcass (156 ␮g/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.
14
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.
Urine
Intravenous administration
Non-cannulated ratsa
(n ⫽ 5)
Cannulated ratsb
(n ⫽ 3)
Topical application
Non-cannulated ratsc
Cannulated ratsc
(n ⫽ 4)
Bile
85.2 ⫾ 2.3
50.8 ⫾ 1.3*
44.5 ⫾ 1
17.3 ⫾ 1.8
(n ⫽ 13)
18.6 ⫾ 0.4
6.7 ⫾ 0.4
* Significant difference from non-cannulated rats dosed in the same conditions ( p ⬍ 0.05).
a
Values obtained 72 h after a single intravenous administration of DBP (1 mg/kg).
b
Values obtained 30 h after a single intravenous administration of DBP (1 mg/kg).
c
Excretion in urine or feces 24 h after a topical administration of neat DBP (10 ␮l/cm2, 10 cm2).
d
Area under the plasma time curve of the radioactivity was calculated from the plasma concentrations reported in the Table 3.
Feces
Plasma (AUC)
9.3 ⫾ 1.0
1.82 ⫾ 0.06
0.1 ⫾ 0.0*
1.20 ⫾ 0.31*
1.5 ⫾ 0.2
(n ⫽ 13)
0.12d
0.0 ⫾ 0.0*
0.11 ⫾ 0.3
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 7. Time course of plasma concentration and biliary excretion rates of total
radioactivity after a single i.v. administration of [14C]DBP in haired male rats.
852
PAYAN ET AL.
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 ␮l/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.
Haired Female Rats
(n ⫽ 4)
Hairless Male Rats
(n ⫽ 6)
38.1 ⫾ 1.7
0.3 ⫾ 0.1
1.4 ⫾ 0.2
12.1 ⫾ 0.7
13.7 ⫾ 0.8
10.5 ⫾ 0.8
4.0 ⫾ 0.5
13.1 ⫾ 1.8
5.8 ⫾ 0.7
0.8 ⫾ 0.2
7.3 ⫾ 0.9
21.2 ⫾ 1.7
90.3 ⫾ 1.0
61.1 ⫾ 1.5
69.1 ⫾ 1.5
0.0036 ⫾ 0.0007
0.28 ⫾ 0.02
0.60 ⫾ 0.08
0.012 ⫾ 0.02
0.08 ⫾ 0.02
0.27 ⫾ 0.04
0.17 ⫾ 0.01
34.0 ⫾ 5.2
0.4 ⫾ 0.2
1.8 ⫾ 0.5
10.3 ⫾ 1.7
12.6 ⫾ 3.1
8.9 ⫾ 1.1
1.3 ⫾ 0.7*
17.3 ⫾ 2.7
3.3 ⫾ 0.7*
1.1 ⫾ 0.2
9.9 ⫾ 3.4
23.7 ⫾ 1.5
90.8 ⫾ 0.5
56.0 ⫾ 4.4
67.0 ⫾ 1.6
0.0034 ⫾ 0.0006
0.37 ⫾ 0.03*
0.61 ⫾ 0.06
0.019 ⫾ 0.004*
0.11 ⫾ 0.03
0.38 ⫾ 0.15
0.17 ⫾ 0.01
57.0 ⫾ 1.8*
0.7 ⫾ 0.1*
3.6 ⫾ 0.7*
30.9 ⫾ 2.7*
18.0 ⫾ 1.2*
3.6 ⫾ 0.3*
9.5 ⫾ 0.6*
1.8 ⫾ 0.3*
2.6 ⫾ 0.4*
0.2 ⫾ 0.0*
0.5 ⫾ 0.1*
25.0 ⫾ 1.7
97.3 ⫾ 0.6*
70.9 ⫾ 2.4*
71.7 ⫾ 2.4
0.001 ⫾ 0.0001*
0.36 ⫾ 0.03*
0.40 ⫾ 0.03*
0.050 ⫾ 0.001
0.02 ⫾ 0.003*
0.02 ⫾ 0.004*
0.22 ⫾ 0.01*
* Significant difference from haired male rats group ( p ⬍ 0.05).
a
Percentage of applied dose.
b
Percentage of the applied dose per ml of plasma.
c
AUC plasma ⫽ % of the applied dose/ml ⫻ h.
d
Kel, elimination rate constant of 14C in plasma (1/h).
e
Percentage of the applied dose per cm2.
f
Gram per cm2 of 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
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 ␮l/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.
Absorbed dosea from
% of applied dose in excreta and carcass
% [Urine(percutaneous)/urine(i.v.)]
% [AUC(percutaneous)/AUC(i.v.)] in plasma
Absorption fluxb from
14
C Concentration in plasma (8 or 24 h)
14
C Urinary excretion rate (8–24 h)
Haired Male Rats
(n ⫽ 9)
Haired Female Rats
(n ⫽ 4)
61.1 ⫾ 1.5
66.4 ⫾ 4.3
29.7 ⫾ 3.7*
56.0 ⫾ 4.4
65.9 ⫾ 11.1
33.5 ⫾ 3.5*
35 ⫾ 4
92 ⫾ 6**
43 ⫾ 4
79 ⫾ 10**
* Significant difference from the value determined with excreta and carcass radioactivity content ( p ⬍ 0.05).
** Significant difference from the absorption flux determined with 14C concentration in plasma ( p ⬍ 0.05).
*** Significant difference from the value obtained in haired male rats ( p ⬍ 0.05).
a
Percentage of the applied dose.
b
␮g/cm2/h.
Hairless Male Rats
(n ⫽ 6)
71.9 ⫾ 2.4***
71.2 ⫾ 1.7
20.9 ⫾ 1.4*
57 ⫾ 5*
237 ⫾ 17**,***
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Urine 0–72 ha
Urine 0–4 h
Urine 4–8 h
Urine 8–24 h
Urine 24–48 h
Urine 48–72 h
Feces 0–72 ha
Carcassa
Cage washinga
Application site skina
Skin around the application sitea
Washing of the application site skina
Recoverya
Percentage of the absorbed dosea
Percentage of the penetrated dosea
Plasmab
AUC0–72 h plasmac
AUC0–inf plasmac
Keld
Application site of skine
Around application site of skine
Weight of skinf
Haired Male Rats
(n ⫽ 9)
853
TOXICOKINETICS OF DBP
FIG. 9. Time course of
14
C in plasma after a topical application of neat
[14C]DBP in rats.
Twenty-four hours after a topical application of neat [14C]DBP (10 ␮l/cm2; 10
cm2), the skin was washed, and the animals were sacrificed 48 h later. Values are
expressed as means ⫾ S.E.M. ●, haired male (n ⫽ 9); ⫻, haired female (n ⫽ 4);
f, hairless male rats (n ⫽ 6). 1 nmol-Eq ⫽ 0.0003% of the applied dose.
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
Values are expressed as means ⫾ S.E.M. (n ⫽ 3 ⫻ 3 rats; 2 cells/rat). The topical
dose was 50 mg/cm2 on a skin area of 1.76 cm2. ⽧, haired male rats; f, hairless
male rats. F1 (␮g-Eq DBP/cm2/h) ⫽ 26.4 ⫾ 0.7; tlag1 (h) ⫽ 8.1 ⫾ 0.9. F2 (␮g-Eq
DBP/cm2/h) ⫽ 38.8 ⫾ 1.1; tlag2 (h) ⫽ 8.3 ⫾ 0.98.
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.
Acknowledgment. We thank M. Roussel for expert secretarial
service.
References
Bando H, Morhi S, Yamashita F, Takakura Y and Hashida M (1997) Effects of skin metabolism
on percutaneous penetration of lipophilic drugs. J Pharm Sci 86:759 –761.
Brandt K (1985) Final report on the safety assessment of dibutyl phthalate, and diethyl phthalate.
J Am Coll Toxicol 4:267–303.
Cater BR, Cook MW, Gangolli SD and Grasso P (1977) Studies on di-butylphthalate induced
testicular atrophy in the rat: effect on zinc metabolism. Toxicol Appl Pharmacol 41:609 – 618.
Elsisi AE, Carter DE and Sipes IG (1989) Dermal absorption of phthalate diesters in rats.
Fundam Appl Toxicol 12:70 –77.
Ema M, Amano H, Itami T and Kawasaki H (1993) Teratogenic evaluation of di-n-butylphthalate
in rats. Toxicol Lett 69:197–203.
Ema M, Amano H and Ogawa Y (1994) Characterization of the developmental toxicity of
di-n-butylphthalate in rats. Toxicology 86:163–174.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 10. Comparison of in vitro cumulative percutaneous absorption of 14C in
haired and hairless male rats after a topical application of neat [14C]DBP.
854
PAYAN ET AL.
Nikoronow M, Mazur H and Piekacz H (1973) Effect of orally administered plasticizers and
polyvinyl chloride stabilizers in the rat. Toxicol Appl Pharmacol 26:253–259.
Oishi S and Hiraga K (1980) Testicular atrophy induced by phthalic acid esters: effect on
testosterone and zinc concentration. Toxicol Appl Pharmacol 53:35– 41.
Peters JW and Cook RM (1973) Effect of phthalate esters on reproduction in rats. Environ Health
Perspect 3:91–94.
Ritschel WA (1980) Handbook of Basic Pharmacokinetics, pp 285–289, Drug Intelligence
Publications, Inc., Hamilton, IL.
Saillenfait AM, Payan JP, Fabry JP, Beydon D, Langonné I, Galissot F and Sabaté JP (1998)
Assessment of the developmental toxicity, metabolism, and placental transfer of di-n-butyl
phthalate administered to pregnant rats. Toxicol Sci 45:212–224.
Scott RC, Dugard PH, Ramsey JD and Rhodes C (1987) In vitro absorption of some o-phthalate
diesters through human and rat skin. Environ Health Perspect 74:223–227.
Shiota K and Nishimura H (1982) Teratogenicity of di(2-ethylhexyl)phthalate (DEHP) and
di-n-butyl phthalate (DBP) in mice. Environ Health Perspect 45:65–70.
Singh AR, Lawrence WH and Autian J (1972) Teratogenicity of phthalate esters in rats. J Pharm
Sci 61:51–55.
Smith CC (1953) Toxicity of butyl stearate, dibutyl sebacate, dibutyl phthalate and methoxyethyl
oleate. Ind Hyg Occup Med 7:310 –318.
Srivastava SP, Srivastava S, Saxena DK, Chandra SV and Seth PK (1990) Testicular effects of
di-n-butyl phthalate (DBP): biochemical and histopathological alterations. Arch Toxicol 64:
148 –152.
Tanaka A, Matsumoto A and Yamaha T (1978) Biochemical studies on phthalic esters. III.
Metabolism of dibutyl phthalate (DBP) in animals. Toxicology 9:109 –123.
Tse FLS, Ballard F and Shaffe JM (1982) A practical method for monitoring drug excretion and
enterohepatic circulation in the rat. J Pharmacol Methods 7:139 –144.
White RD, Carter DE, Earnest D and Mueller J (1980) Absorption and metabolism of three
phthalate diesters by the rat small intestine. Food Cosmet Toxicol 18:383–386.
Williams DT and Blanchfield BJ (1975) The retention, distribution, excretion and metabolism of
dibutylphthalate-7-14C in the rat. J Agric Food Chem 23:854 – 858.
Woodward KN (1988) Phthalate Esters: Toxicity and Metabolism, vols I and II, CRC Press, Inc.,
Boca Raton, FL.
World Health Organization (1997) Environmental health criteria for di-n-butylphthalate, in
Environmental Health Criteria 189.
Yamada A (1974) Toxicity of phthalic acid esters and hepatotoxicity of di-2-ethylhexylphthalate.
J Food Hyg Soc Jpn 15:147–152.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Ema M, Kurosaka R, Amano H and Ogawa Y (1995a) Developmental toxicity evaluation of
mono-n-butylphthalate in rats. Toxicol Lett 78:101–106.
Ema M, Kurosaka R, Harazono A, Amano H and Ogawa Y (1996) Phase specificity of
developmental toxicity after oral administration of mono-n-butylphthalate in rats. Arch Environ Contam Toxicol 31:170 –176.
Ema M, Kurosaka R and Ogawa Y (1995b) Comparative developmental toxicity of n-butyl
benzyl phthalate and di-n-butylphthalate in rats. Arch Environ Contam Toxicol 28:223–228.
Foster PM, Cook MW, Thomas LV, Walters DG and Gangolli SD (1982) Differences in urinary
metabolic profile from di-n-butylphthalate treated rats and hamsters. A possible explanation
for species differences in susceptibility to testicular atrophy. Drug Metab Dispos 11:59 – 61.
Fukuoka M, Tanimoto T, Zhou Y, Kawasaki N, Tanaka A, Ikemoto I and Machida T (1989)
Mechanism of testicular atrophy induced by di-n-butylphthalate in rats. Part 1. J Appl Toxicol
9:277–283.
Gangolli SD (1982) Testicular effects of phthalate esters. Environ Health Perspect 45:77– 84.
Gray TJ, Rowland IR, Foster PM and Gangolli SD (1982) Species differences in the testicular
toxicity of phthalate esters. Toxicol Lett 11:141–147.
Illel B, Schaeffer H, Wepierre J and Doucet O (1991) Follicles play an important role in
percutaneous absorption. J Pharm Sci 80:424 – 427.
Journal of Property Investment and Finance (JPIF) (1995) Production of plastics. Plastics
46:104.
Kaneshima H, Yamaguchi T, Okui T and Naitoh M (1978) Studies on the effects of phthalate
esters on the biological system (Part 2): in vitro metabolism and biliary excretion of phthalate
esters in rats. Bull Environ Contam Toxicol 19:502–509.
Kao J, Hall J and Helman G (1988) In vitro percutaneous absorption in mouse skin influence of
skin appendages. Toxicol Appl Pharmacol 94:93–103.
Lehman AJ (1955) Insect repellents. Assoc Food Drug Off 19:87–99.
Mint A and Hotchkiss SA (1993) Percutaneous absorption of dimethylphthalate and di-nbutylphthalate through rat and human skin in vitro, in Prediction of Percutaneous Penetration
(Brain KR, James VJ, Hadgraft J and Walters KA eds) vol 3B, pp 646 – 657, STS Publishing,
Cardiff.
Mint A, Hotchkiss SA and Caldwell J (1994) Percutaneous absorption of diethylphthalate
through rat and human skin in vitro. Toxicol In Vitro 2:251–256.
National Toxicology Program (1995) Technical Report on Toxicity Studies of Di-Butylphthalate
(CAS No. 84-74-2) Administered in Feed to F344/N Rats and B6C3 Mice. Toxicity Series no.
30. United States Department of Health and Human Services, Research Triangle Park, North
Carolina.