TOXICOLOGICAL SCIENCES 91(2), 341–355 (2006)
doi:10.1093/toxsci/kfj160
Advance Access publication March 16, 2006
Absorption, Distribution, Metabolism, and Elimination of 8-2
Fluorotelomer Alcohol in the Rat
William J. Fasano,*,1 Stephen C. Carpenter,* Shawn A. Gannon,* Timothy A. Snow,* Judith C. Stadler,*
Gerald L. Kennedy,* Robert C. Buck,† Stephen H. Korzeniowski,† Paul M. Hinderliter,‡ and Raymond A. Kemper§
*DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware 19714; †DuPont Chemical Solutions Enterprise, Wilmington,
Delaware 19880; ‡Battelle Pacific Northwest, Richland, Washington 99352; and §Boehringer-Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877
Received January 15, 2006; accepted March 3, 2006
The absorption, distribution, metabolism, and elimination
of [3-14C] 8-2 fluorotelomer alcohol (8-2 FTOH, C7F1514CF2CH2CH2OH) following a single oral dose at 5 and 125 mg/kg in
male and female rats have been determined. Following oral
dosing, the maximum concentration of 8-2 FTOH in plasma
occurred by 1 h postdose and cleared rapidly with a half-life of
less than 5 h. The internal dose to 8-2 FTOH, as measured by area
under the concentration-time curve to infinity, was similar for
male and female rats and was observed to increase in a dosedependent fashion. The majority of the 14C 8-2 FTOH ( > 70%)
was excreted in feces, and 37–55% was identified as parent. Less
than 4% of the administered dose was excreted in urine, which
contained low concentrations of perfluorooctanoate (~ 1% of total
14
C). Metabolites identified in bile were principally composed of
glucuronide and glutathione conjugates, and perfluorohexanoate
was identified in excreta and plasma, demonstrating the metabolism of the parent FTOH by sequential removal of multiple CF2
groups. At 7 days postdose, 4–7% of the administered radioactivity was present in tissues, and for the majority, 14C concentrations
were greater than whole blood with the highest concentration in
fat, liver, thyroid, and adrenals. Distribution and excretion of
a single 125-mg/kg [3-14C] 8-2 FTOH dermal dose following a 6-h
exposure in rats was also determined. The majority of the dermal
dose either volatilized from the skin (37%) or was removed by
washing (29%). Following a 6-h dermal exposure and a 7-day
collection period, excretion of total radioactivity via urine ( <
0.1%) and feces ( < 0.2%) was minor, and radioactivity concentrations in most tissues were below the limit of detection. Systemic availability of 8-2 FTOH following dermal exposure was
negligible.
Key Words: 8-2 fluorotelomer alcohol; absorption, distribution,
metabolism, and elimination (ADME); glutathione conjugates;
perfluorinated carboxylic acids; perfluorooctanoate; peroxisome
proliferation; pharmacokinetics.
1
To whom correspondence should be addressed. Fax: (302) 366-5207.
E-mail: [email protected].
The presence of perfluorosulfonic acids and perfluorocarboxylic acids (PFCAs) in mammals and the environment has
fueled a growing interest into the sources and biological fate of
this class of chemistry (Ellis et al., 2003, 2004; Moody et al.,
2002; Olsen et al., 2003; Verreault et al., 2005). Recently, the
potential sources and fate of PFCAs have been discussed
(Prevedouros et al., 2005). Fluorotelomer-based substances
have been proposed as a possible indirect source for PFCAs,
since they may contain PFCAs as an unintended reaction
byproduct and may also degrade to form PFCAs. Fluorotelomer alcohols (FTOHs) are a raw material comprising an
even number of fluorinated carbons appended to an ethanol
moiety (F(CF2)xC2H4OH; x ¼ 6, 8, 10) and are used in the
manufacture of products with unique surface properties used in
a variety of industrial and consumer applications (Baker and
Rao, 1994; Kissa, 2001). 8-2 FTOH is the homologue produced
in the largest volume. In 2000–2002, global production of
perfluoroalkyl iodide, which is used to make fluorotelomerbased products, exceeded 5000 metric tons (Telomer Research
Program, 2002).
The subchronic and developmental/reproductive toxicities
of 8-2 FTOH (DuPont-9478, unpublished; Mylchreest et al.,
2005b) and of a commercial mixture containing 6-2, 8-2, and
10-2 FTOH (Ladics et al., 2005; Mylchreest et al., 2005a) have
been reported. In the subchronic 90-day oral study, where 8-2
FTOH was administered at 1, 5, 25, and 125 mg/kg/day, the no
observable effect level for male and female rats was 5 and
25 mg/kg/day, respectively, and was based on liver necrosis
and nephropathy (DuPont-9478, unpublished). Hepatic boxidation, a measure of peroxisome proliferation, was increased
in females at 25 mg/kg/day and in males and females at 125 mg/
kg/day; plasma and urine fluoride concentrations, as measured
using a fluoride-selective electrode, were significantly increased
in males and females at the 125-mg/kg/day dose level. The
developmental toxicity study in rats reported that 8-2 FTOH
was not a selective developmental toxin (Mylchreest et al.,
2005b).
The metabolism of 8-2 FTOH in male rats following a 400mg/kg single oral dose and analysis of plasma at 6 h postdose
The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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342
FASANO ET AL.
have been reported (Hagen et al., 1981). Perfluorooctanoate
(PFO), the disassociated form of the free acid, 8-2 fluorotelomer acid (CF3(CF2)6CF2CH2COOH; 8-2 FTA), and 8-2
fluorotelomer unsaturated acid (CF3(CF2)6CF¼CHCOOH; 8-2
FTUA) were identified in plasma. More recently, Kudo et al.
(2005) examined the effects of 8-2 FTOH dietary intake in
mice at 0, 0.025, 0.05, 0.1, and 0.2% (wt/wt) and reported
evidence of a dose- and duration-dependent liver enlargement
caused by peroxisome proliferation. PFO, perfluorononanoate
(PFN), 8-2 FTA, and two unidentified metabolites were
observed in liver and plasma.
The metabolic fate of 8-2 FTOH in rat hepatocytes has been
reported to occur primarily by direct conjugation of parent
to form O-glucuronide and O-sulfate (Martin et al., 2005). A
transient 8-2 fluorotelomer aldehyde (CF3(CF2)7CH2CH¼O;
8-2 FTAL) and the presence of 8-2 FTA, 8-2 FTUA, 7-3 Acid
(CF3(CF2)6CH2CH2COOH), unsaturated 7-3 Acid (CF3(CF2)6CH¼
CHCOOH; 7-3 uAcid), and glutathione (GSH) conjugates of the
8-2 FTOH unsaturated aldehyde (8-2 uFTALGS), 8-2 FTUA
(8-2 FTUAGS), and the reduced form the 8-2 uFTALGS
(8-2uFTOHGS) were also observed. Martin et al. (2005) also
reported results from a single ip 400-mg/kg dose to male rats
and analysis of plasma at 6 h postdose, confirming the observation of PFO, 8-2 FTA, and 8-2 FTUA by Hagen et al. (1981).
The metabolism of 8-2 FTOH in sludge, soils, and groundwater sediment has been studied to determine whether and
to what extent PFCAs, including PFO, would be formed
(Dinglasan et al., 2004; Wang et al., 2005a,b). These studies
report that the relative amount of PFO produced from 8-2
FTOH metabolism was minor (less than 6%), suggesting that
microbial metabolism of 8-2 FTOH is unlikely a principal
source for PFO in the environment. A detailed microbial
metabolic pathway for 8-2 FTOH has been proposed (Wang
et al., 2005b).
Thus far, 8-2 FTOH in vivo rat metabolism experiments have
been limited in scope, generally qualitative, and lacking
a quantitative measure of absorption, distribution, metabolism,
and elimination (ADME) and an answer to the fundamental
question of how much PFO is produced. The goal of the present
research was to quantitatively determine 8-2 FTOH ADME in
male and female rats following a single oral dose and also the
absorption, distribution, and excretion following a 6-h dermal
exposure in the rat utilizing radiolabeled 8-2 FTOH (DuPont6997, unpublished).
MATERIALS AND METHODS
Chemicals. The test material, 1H,1H,2H,2H-perfluorodecanol (CF3(CF2)7
CH2CH2OH; 8-2 FTOH; CASN 678-39-7), was supplied by E.I. du Pont de
Nemours and Company (Wilmington, DE). The sample had a purity of 99.2%.
The primary impurity (0.8%) was unsaturated 8-2 FTOH (CF3(CF2)6CF¼
CHCH2OH; 8-2 uFTOH). 8-2 FTOH is relatively insoluble in water (148 lg/l)
and has a vapor pressure of 3 Pa (~ 0.02 mm Hg) at 22C (Krusic et al., 2005).
Radiolabeled [3-14C] 8-2 FTOH was synthesized by Perkin-Elmer Life and
Analytical Sciences, Inc. (Boston, MA). The radiolabeled test material had
a specific activity of 54.3 mCi/mmol and a radiochemical purity of > 98%. The
material was stored at 70C.
8-2 Fluorotelomer acid (CF3(CF2)7CH2COOH; 8-2 FTA) and 8-2 FTUA,
were provided by E.I. du Pont de Nemours and Company. The PFCAs,
perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, and
perfluorononanoic acid were purchased from Sigma Aldrich (St. Louis, MO),
and their purity was > 95%.
Media and reagents for the hepatocyte isolations were obtained from
Invitrogen (Carlsbad, CA).
Animals. Male and female Crl:CD (SD)IGS BR rats, including jugular
vein– and bile duct–cannulated animals, were obtained from Charles River
Laboratories, Inc. (Raleigh, NC). At the time of dosing, rats were approximately 6–8 weeks of age.
Oral dosing. Three main oral gavage–dosing experiments were conducted:
material balance, biliary elimination, and plasma kinetics. Four male and four
female rats per dose level per experiment were given single oral gavage doses
formulated with [3-14C] 8-2 FTOH at 5 and 125 mg/kg. The dose levels were
selected to match the dose levels used in the rat subchronic toxicity study
(DuPont-9478, unpublished). Methylcellulose (0.5% wt/vol) was selected as
the vehicle for consistency with the 90-day subchronic study and to facilitate
comparisons between oral and dermal exposure routes. All rats were fasted for
approximately 16 h prior to oral dosing. Following dosing, rats for the material
balance experiment were housed in glass metabolism units, and excreta was
collected on dry ice at 6, 12, and 24 h and at 24-h intervals thereafter through
168 h. At the end of the collection period, rats were anesthetized by exposure to
carbon dioxide and sacrificed by exsanguination via cardiac puncture. Selected
tissues were collected for analysis of total radioactivity.
Rats for the biliary elimination experiment were received from the supplier
with a cannula surgically implanted in the bile duct. Rats were housed one per
metabolism unit and provided with isotonic dextrose/KCl/NaCl as the sole
source of drinking fluid for the duration of the in-life phase. Bile was collected
on dry ice at approximately 6, 12, and 24 h and every 24 h until sacrifice (120 h
postdose).
Rats for the plasma kinetic experiment were received from the supplier with
a cannula inserted in the jugular vein. Following fasting, each rat received a
single oral dose formulated with [3-14C] 8-2 FTOH. Following dosing, serial
blood samples were collected from the jugular vein cannula, and alternatively
from the orbital sinus or lateral tail vein and placed into tubes containing
EDTA. Whole-blood samples were collected predose and at approximately 5,
15, and 30 min and 1, 2, 4, 6, 12, and 24 h postdose and at 24-h intervals
thereafter until 168 h. Serial whole-blood samples were kept on wet ice, and
plasma was separated from the red cell fraction by centrifugation. Plasma was
stored frozen at < 10C prior to analysis.
In a separate (supplemental) experiment, a single male and female rat were
administered a 125-mg/kg single oral dose of 8-2 FTOH fortified with [3-14C]
8-2 FTOH and serial blood samples collected at 1, 2, 4, 8, 24, and 48 h. Plasma
was separated from the red cell fraction by centrifugation and pooled (1–24 h)
for metabolite profiling and identification.
Dermal exposure. The study comprised a single exposure group of three
male and three female rats. 8-2 FTOH fortified with [3-14C] 8-2 FTOH was
administered at a dose rate of 125 mg/kg in 0.5% methylcellulose. On the day
prior to dosing, the back and shoulder area were clipped free of hair and washed
with a 2% Ivory soap solution in water. Following shaving and washing, a glass
O-ring with an internal exposure area of approximately 5.3 cm2 was glued to
the shaved skin with Scotch Brand Crazy Glue Gel and the area covered with
gauze and wrapped with Coban. Rats were fitted with cervical collars and
acclimated overnight prior to dosing. At dosing, rats were anesthetized with
Isoflurane, and the dermal dose was applied at a rate of approximately 10 ll/cm2
to the area within the glass O-ring. Immediately following administration
of the dose, the dose site was covered with an airtight, organic volatile trap
containing untreated, granular activated charcoal, size 8–20 mesh (Sigma
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
Chemical Company, St. Louis, MO). At the conclusion of the 6-h exposure
period, the charcoal trap was removed and immediately placed into a glass
container with ethanol, and the dose site was washed with natural sponge
soaked in ethanol (use of a 2% soap and water solution had previously been
shown to result in low recovery of radiolabeled 8-2 FTOH). Rats were then
fitted with a fresh charcoal trap and body wrap and returned to their metabolism
units. At 24 h postdose, the charcoal trap was again collected, placed into
ethanol, and a fresh charcoal trap applied, which remained in place until
termination at 168 h postdose. Urine and feces were collected on dry ice at
approximately 6 (end of exposure), 12, and 24 h following dosing and at 24-h
intervals thereafter until sacrifice at 168 h. At sacrifice, rats were anesthetized by exposure to carbon dioxide, exsanguinated, and tissues collected for
analysis.
Rat hepatocyte in vitro metabolism. In order to support the identification
of metabolites from the in vivo experiments, 8-2 FTOH was incubated with
isolated rat hepatocytes. Rat hepatocytes were prepared by a modification of the
method from Seglen (1976) with a two-stage collagenase perfusion using
commercially available reagents. The isolated cells were diluted to a final
concentration of approximately 5 3 106 cells/ml, and [3-14C] 8-2 FTOH was
added to a concentration of 50lM in a final volume of 1 ml. The sample was
incubated at 37C for 2 h and terminated by the addition of acetonitrile (vol/
vol). The sample was filtered (0.45 lm) and used for metabolite identification.
Sample collection, processing, and 14C analysis. Quantitative analysis of
samples from the oral (material balance, plasma kinetic, and biliary elimination) and dermal experiments was performed by taking representative aliquots,
at least in duplicate or in triplicate when sufficient sample was available, and
analyzing by either direct analysis (i.e., liquid samples) using liquid scintillation counting (LSC) or by combustion analysis (i.e., semisolid and solid
samples) using a Packard Tri-Carb Automatic Sample Oxidizer followed by
LSC using Packard Tricarb 2500TR liquid scintillation counter (Meriden, CT).
The application skin site and nondosed skin samples from the dermal
exposure experiment were digested in Soluene-350 (Perkin-Elmer Life and
Analytical Sciences, Inc.) and analyzed for total radioactivity by LSC.
Quantitative analysis of plasma for 8-2 FTOH. Plasma was thawed at
room temperature and a 50-ll aliquot removed with an airtight syringe. The
sample was injected into a 2-ml glass vial insert, heated to approximately 50C
for 10 min, and the headspace extracted for 5 min using a 100-lm film
thickness polydimethylsiloxane solid-phase microextraction fiber. Following
extraction, the fiber was desorbed at 250C for 1 min and analyzed using an
Agilent Gas Chromatography (GC) HP 6890 Series/Mass Selective Detection
(Agilent Technologies, Palo Alto, CA) with a Gerstel MultiPurpose Sampler
(Gerstel Inc., Baltimore, MD). Separation was achieved using a DB-5MS
Capillary Column, 30 m 3 0.250 mm 3 1.0 lm (J&W Scientific, Folsom, CA)
and an inlet program temperature of 250C at 12.5 pounds per square inch (psi)
with a total flow of 81 ml/min operated in splitless mode. The mass
spectrometer (MS) detector was operated in a single-ion monitoring mode
with source temperature of 250C.
Quantitative analysis of plasma for PFO and 8-2 FTA. Plasma was
thawed at room temperature and a 50-ll aliquot applied to an Isolute array
protein precipitation plate (Jones Chromatography, Lakewood, CO) followed
by the addition of acetonitrile to precipitate plasma proteins. The column was
eluted under vacuum, and the extracts were analyzed using a Waters Alliance
2795 Liquid Chromatograph (LC) coupled to a Micromass Quattro micro triple
quadrupole MS equipped with a MASSLYNX data acquisition system
(Micromass Inc., Manchester, United Kingdom). LC separation was achieved
using a Phenomenex Synergi Max-RP, 2.0 3 20 mm, 2-lm particles at 35C,
and mobile phase of 50mM ammonium acetate and acetonitrile at a flow rate of
0.25 ml/min. The MS was operated in the negative ionization mode with a
capillary voltage of 2.8 kV, a cone voltage of 15 V, and a source temperature of
130C. The analytes of interest were quantitated in multiple response–
monitoring mode with transitions of m/z 413/369 for PFO and 477/393
for 8-2 FTA.
343
Urine sample preparation and metabolite identification. Urine samples
from rats in the oral dose experiments were pooled through 90% of the total radioactive dose eliminated over the 168-h collection period and filtered (0.45 lm),
and representative samples were analyzed by liquid chromatography–accurate
radioisotope counting (LC-ARC) and LC-MS.
Feces sample preparation and metabolite identification. Feces samples
from rats in the oral dose experiment were pooled through 90% of the total
radioactive dose eliminated via the feces, and aliquots solvent was extracted
using an accelerated solvent extraction system (Dionex Corporation, Sunnyvale, CA) with repeat cycles of 75:25 acetonitrile:water and 75:25 methanol:water at 100C and 1500–2000 psi. The final extract was filtered (0.45 mm) and
analyzed by LC-ARC and LC-MS. Extraction of radioactivity exceeded 80%.
Bile sample preparation and metabolite identification. Bile samples from
rats in the biliary elimination experiment were pooled through 90% of the
total radioactive dose eliminated via the bile, and samples (200 ll) were filtered
(0.45 lm) and mixed with 2M potassium chloride and 1M ammonium acetate.
The sample was then partitioned with hexane, the aqueous phase isolated, and a
selected representative sample was submitted for LC-ARC and LC-MS analysis.
Plasma sample preparation and metabolite identification. Serial plasma
samples collected from a single male rat (supplemental experiment) at 1, 2, 4,
8, and 24 h were pooled, precipitated with acetonitrile, centrifuged, filtered
(0.45 lm), and submitted for LC-MS analysis.
Liquid chromatography–accurate radioisotope counting. Representative
samples of urine, feces extract, and bile extract were quantitatively analyzed
using a Hewlett-Packard 1100 Series ChemStation. Separation was achieved
using a FluroFlash 4.6 mm 3 150 mm column (Fluorous Technologies,
Pittsburgh, PA) and a gradient of 0.15% acetic acid in water and methanol with
a flow rate of 1 ml/min (ambient temperature). Column effluent was mixed with
StopFlow AQ, High Efficiency Liquid Scintillation Cocktail (AIM Research
Co., Newark, DE), followed by radiodetection using a Radiomatic 150TR
Series FLO-One System/LC-ARC system (AIM Research Co.), containing a
1300-ll flow cell, stopping effluent flow in the liquid cell every 10 s, with a hold
time for counting of 2 min. For each injection, the regions of interest were
identified and integrated. Based on the total radioactivity injected on column,
the percentage of total dose administered represented by each integrated region
of interest was determined (percentage of dose). Recovery of the on-column
injection of radioactivity was > 90%.
Individual metabolites (regions of interest identified by LC-ARC) from
urine, feces, and bile were isolated by fraction collection in a repeat LC run and
the isolated fractions submitted for mass spectral analysis.
Liquid chromatography–mass spectral analysis. Samples of urine (both
neat samples and fractions representing isolated peaks of interest), feces
extract, bile extract, plasma extract, and hepatocyte extract solution, along with
matrix control samples were screened using a Waters Alliance 2790 LC
coupled to a Micromass QToF II hybrid quadrupole/time-of-flight MS, operated in negative electrospray ionization mode, equipped with a MASSLYNX
data acquisition system (Micromass Inc.). Separation was achieved using a
Zorbax SB-C18 RP, 2.1 3 150 mm, 5-lm particle size, a gradient mobile phase
of 0.15% acetic acid and methanol (urine only) and 2mM ammonium acetate
and methanol, at a flow rate of 0.3 ml/min. The MS system was operated with
a capillary voltage of 0.8–2.9 kV, a cone voltage of 16–24 V, a source
temperature of 125C, and a desolvation temperature of 350C.
Data presentation and statistical analyses. The calculated values presented in the tables for the oral and dermal material balance experiments were
generated by Debra v.5.1a (LabLogic Systems Ltd., Sheffield, United
Kingdom). The statistical significance of selected data was assessed by the
Student’s t-test using Microcal, version 7.0 (OriginLab Corporation, Northampton, MA). A p value of 0.05 was considered significant.
Noncompartmental pharmacokinetic parameters for plasma total radioactivity, 8-2 FTOH, PFO, and 8-2 FTA were generated using WinNonlin ver. 4.0,
Pharsight Corporation (Mountain View, CA). Calculated parameters included
344
FASANO ET AL.
time to maximum plasma concentration (Tmax) in hours; maximum plasma
concentration observed (Cmax) in ng equiv/ml (for total radioactivity) or ng/ml
(for 8-2 FTOH, PFO, and 8-2 FTA); area under the concentration-time curve to
infinity (AUCinf) in hng equiv/ml (for total radioactivity), and hng/ml (for 8-2
FTOH, PFO, and 8-2 FTA); and the terminal elimination half-life (t1/2) in hours.
Plasma PFO concentration data after 48 h for female rats at the 125-mg/kg
dose level were excluded due to questionable results. The lowest standard
concentration for determining the plasma concentrations of 8-2 FTOH, PFO,
and 8-2 FTA was 0.005 lg/ml (5 ng/ml) and was assumed to be equal to the
limit of quantitation (LOQ).
RESULTS
Oral Dosing, Material Balance, and Biliary Elimination
Following a single oral dose of [3-14C] 8-2 FTOH to male and
female rats at 5 and 125 mg/kg and a 168-h collection period, the
majority of the administered radioactivity (> 70%) was excreted
in the feces, exclusive of sex and dose level (Table 1). Excretion
of radioactivity via the urine was a minor pathway, with approximately 2.1- to 2.4-fold more total radioactivity recovered in
female urine (2.7–3.6%) compared to male urine (1.3–1.5%).
Recovery of the administered radioactive dose was > 76%.
Following a 5-mg/kg single oral dose of [3-14C] 8-2 FTOH,
39 and 45% of the dose was eliminated via the bile in male and
female rats, respectively, as compared to 21 and 19% at the
125-mg/kg dose level. Total systemic absorption of 8-2 FTOH
(calculated as the sum of the percent administered 14C dose
recovered in urine, tissues, and bile) was estimated to be 49 and
57% at 5 mg/kg, and 27 and 27% at 125 mg/kg for male and
female rats, respectively.
Owing to the high amount of radioactivity eliminated via the
feces, the semivolatile character of 8-2 FTOH, and its low
miscibility in water (Krusic et al., 2005), it was hypothesized
TABLE 1
Material Balance at 168 h following a Single Oral Dose of
[3-14C] 8-2 FTOH to Male and Female Rats (n ¼ 4 per sex)
Percentage of
14
C administered dose
5 mg/kg
Males
Mean
Urine
Feces
Bile
Cage wash
Residual feed
Tissues
Total recoveredb
Total absorbedc
SD
1.53a 0.30
76.8
3.72
39.4
9.88
0.55
0.47
0.61
0.72
7.24
0.64
86.8
2.51
49.3
10.5
125 mg/kg
Females
Males
Females
Mean
SD
Mean
SD
Mean
SD
3.61
77.5
45.1
1.27
0.46
6.45
89.3
56.9
1.51
5.44
6.44
0.48
0.41
1.05
5.15
8.23
1.28a
70.3
20.9
0.29a
0.10
4.36
76.3
26.9
0.27
4.41
7.42
0.03
0.02
1.17
4.19
8.10
2.73 0.47
69.1 11.0
18.6
5.55
0.61 0.14
0.09 0.05
4.45 0.95
77.0 11.1
26.5
5.03
that by allowing the feces samples to thaw at room temperature
for processing and blending with water prior to sample
oxidation, a portion of the radioactive 8-2 FTOH volatilized
and was lost, which resulted in the low recovery of radiolabeled
8-2 FTOH from rats in the high (and likely the low) oral dose
groups. To test this hypothesis an additional rat per sex was
dosed at 125 mg/kg, and the feces was maintained frozen on
dry ice and oxidized completely to minimize the potential loss
of radioactivity. The results of this experiment showed an
increase in the amount of radioactivity recovered in the feces
(~ 5–13%) with an overall recovery of > 90%, confirming the
hypothesis.
Overall, and for the vast majority of tissues, the mean
concentration of radioactivity was greater than whole blood at
168 h following an oral dose of [3-14C] 8-2 FTOH (Table 2).
For both male and female rats, fat, liver, adrenals, and thyroid
contained the greatest 14C concentrations at terminal sacrifice.
TABLE 2
Concentration of Radioactivity in Selected Tissues at 168 h
following a Single Oral Dose of [3-14C] 8-2 FTOH to Male
and Female Rats (n ¼ 4 per sex)
Microgram equivalent of 8-2 FTOH per gram of tissue
5 mg/kg
Males
Teeth
Carcass
Skin
Whole blood
Bone marrow
Brain
Fat
Rbc
Heart
Lungs
Spleen
Liver
Kidney
GI tract
GI contents
Thyroid
Thymus
Testes
Ovaries
Pancreas
Adrenals
Plasma
Uterus
Muscle
Bone
125 mg/kg
Females
Mean
SD
Mean
0.04a
0.25
0.27
0.10a
0.14
0.01
1.11a
0.03a
0.09
0.31
0.06
0.77a
0.23
0.28a
0.04
0.47
0.19
0.06
NA
0.16
0.77
0.16a
NA
0.06
0.11
0.02 < LOD
0.02
0.26
0.08
0.21
0.01
0.05
0.09
0.27
0.01
0.01
0.29
2.53
0.00
0.07
0.01
0.09
0.05
0.26
0.02
0.05
0.08
0.49
0.03
0.27
0.06
0.38
0.01
0.08
0.20
0.49
0.09
0.22
0.01
NA
0.26
0.05
0.16
0.57
0.69
0.02
0.04
0.06
0.03
0.05
0.07
0.06
SD
Males
Mean
NA < LOD
0.05
5.06
0.14
5.64
0.01
2.18a
0.15
1.13a
0.01
0.32
0.31 30.6a
0.01
1.27a
0.02
1.92
0.05
6.19
0.01
1.06
0.07 17.1a
0.03
5.15
0.04
3.59
0.05
0.65
0.18 16.1
0.06
3.69
0.96
0.04
NA
0.04
5.89
0.57 14.8
0.01
3.47a
0.02
NA
0.02
1.05
0.02
0.85
Females
SD
Mean
SD
NA < LOD
1.37
6.40
2.57 10.8
0.47
0.79
0.46
5.01
0.07
0.26
8.04 47.0
0.33
0.72
0.93
2.55
1.39
5.53
0.35
1.14
3.34
9.35
1.53
5.25
0.83
6.57
0.18
0.70
13.4
11.6
1.79
5.59
0.18
NA
7.37
4.36
3.30
13.2
13.7
0.86
0.86
1.75
0.86
0.74
0.35
1.29
NA
1.36
3.69
0.12
1.39
0.05
7.53
0.13
0.95
1.02
0.53
1.55
2.19
2.45
0.15
4.52
3.33
2.60
0.91
4.94
0.15
1.77
0.23
0.68
a
Males and females statistically different, p 0.05.
Sum of urine, feces, cage wash, residual feed, and tissues.
c
Sum of urine, bile, cage wash, residual feed, and tissues.
b
Note. < LOD ¼ sample below limit of detection; NA ¼ not applicable.
a
Males and females statistically different, p 0.05.
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
Oral Dosing and Plasma Kinetics
Following oral dosing, total radioactivity, parent 8-2 FTOH,
and each of the quantitated metabolites appeared rapidly in the
plasma of both sexes and at both dose levels (Fig. 1). Tmax was
3.8 h for all metabolites except PFO (Table 3). Cmax for total
radioactivity, the parent telomer alcohol, and each analyte by
dose level were comparable between the sexes (less than twoto threefold difference), with the exception of 8-2 FTA at the
low dose, which was approximately fivefold higher in female
rats. Clearance of 8-2 FTOH and 8-2 FTA was rapid in both
sexes and at both dose levels as indicated by the relatively short
plasma t1/2 of approximately 1–5 h for 8-2 FTOH and 4–18 h
for 8-2 FTA. The internal dose to parent 8-2 FTOH, as measured by AUCinf, was similar for male and female rats by dose
level (less than or equal to twofold difference), which increased
in a dose-dependent fashion for both sexes. However, in male
rats, there was less than a 15-fold increase in AUCinf with a 25fold increase in dose. For PFO, several possibly erroneous
plasma concentrations after 48 h made the kinetic parameter
calculations more difficult to resolve. PFO plasma concentrations in the high-dose females dropped to below the LOQ
345
after 24 h. However, several later time points actually had
quantifiable concentrations of PFO. The reason for this is unclear,
but with the majority of plasma PFO concentrations < LOQ after
24 h, all concentrations were assumed to be < LOQ after 48 h
for the calculation of pharmacokinetic parameters.
The t1/2 for PFO was longer in males (112–217 h) than in
female rats (5.6–17.5 h) at both dose levels. At the 125-mg/kg
dose level, the t1/2 for PFO in males was calculated to be 112 h.
Nondetectable concentrations were ignored at the 120-h time
point, lessening confidence in this value. Internal dose for PFO
was correspondingly higher in males at both dose levels consistent with previous PFO pharmacokinetic studies (Kemper
and Jepson, 2003; Vanden Heuvel et al., 1991). With the
exception of the PFO t1/2 for male rats at the low-dose level
(217 h), the PFO t1/2 was less than the t1/2 for total radioactivity,
indicating that another metabolite (or metabolites) present in
the plasma but not quantitated was responsible for the total
radioactivity terminal elimination half-life.
Total radioactivity internal exposure and elimination were
significantly different from parent 8-2 FTOH. On average, the
total radioactivity AUCinf was 580- to 680-fold and 170- to
200-fold higher than the 8-2 FTOH AUCinf for male and female
rats, respectively. Exclusive of sex, 8-2 FTOH t1/2 was < 5 h
while t1/2 for total radioactivity was much longer (68–216 h).
The total radioactivity AUCinf was much greater than the sum
of the AUCinf values for the measured metabolites in the
plasma, suggesting that another metabolite or metabolites were
present in the plasma. This disparity, when represented as a
percentage of the total radioactivity AUCinf at each dose level
(sum of the individual analyte AUCinf values divided by the
total radioactivity AUCinf 3 100), was greater for females (3.4–
9.4%) than males (25–26%) primarily due to the correspondingly higher concentrations of PFO in the plasma of male rats.
Dermal Exposure
FIG. 1. Plasma concentration-time course of total radioactivity (lg equiv/
ml; diamond symbol), 8-2 FTOH (square), PFO (triangle), and 8-2 FTA (circle)
following a 5-mg/kg (open symbols) and 125-mg/kg (filled symbols) single oral
dose of [3-14C] 8-2 FTOH in (A) male and (B) female rats.
Following a 6-h dermal exposure to [3-14C] 8-2 FTOH at
125 mg/kg and a collection period of 168 h, excretion via urine
(< 0.1%) and feces (< 0.2%) was minor, indicating that
systemic absorption of 8-2 FTOH from topical administration
was negligible (Table 4). The majority of the applied radioactive dose for both male and female rats was either retained in
the organic volatile trap (> 37%) or was washed from the
application skin site (> 29%). Total recovery of the 14C dose
was > 68%. On average, tissues taken at terminal sacrifice did
not contain detectable concentrations of radioactivity (< LOD),
and this was with few exceptions. Based on the average amount
of 8-2 FTOH applied (~ 31,250 lg), total radioactive residues
at terminal sacrifice were detected at the site of application
(21–58 lg equiv/g), the fat (0.25–0.36 lg equiv/g), and liver
(0.09–0.24 lg equiv/g). The total absorbed dose following a
single 6-h dermal exposure and a 7-day recovery collection
period was low ranging from 0.5 to 1% and was primarily due
to the radioactivity that remained at the application site.
346
FASANO ET AL.
TABLE 3
Plasma Kinetic Parameters for Male and Female Rats following a Single Oral Dose of [3-14C] 8-2 FTOH
(mean of n ¼ 4 rats per sex with the SD in brackets)
Total radioactivity
5 mg/kg
Tmax (h)
Cmax (ng/ml)
AUCinf (hng/ml)
t1/2 (h)
125 mg/kg
Tmax (h)
Cmax (ng/ml)
AUCinf (hng/ml)
t1/2 (h)
8-2 FTOH
PFO
8-2 FTA
Male
Female
Male
Female
Male
Female
Male
Female
1.0 (0.0)a
1370 (380)a
0.6 (0.3)a
1140 (210)a
1.0 (0.0)
63 (18)
0.9 (0.3)
50 (35)
42 (36)
81 (14)
7.6 3 104
(1.5 3 104)a
101 (23)a
2.4 3 104
(1.2 3 103)a
70 (10)a
1.3 3 102
(0.37 3 102)
0.9 (0.2)
1.4 3 102
(1.0 3 103)
1.6 (0.7)
2.6 3 104
(1.8 3 104)
217 (110)
1.0 (0.0)
117 (61)
4.4 3 102
(1.5 3 102)
17.5 (24)
0.9 (0.3)
170 (102)
6.0 3 102
(3.5 3 102)
15.2 (15.4)
1.0 (0.0)
852 (198)
1.7 3 103
(0.45 3 103)
4.6 (3.6)
1.8 (0.5)
11,300 (1600)b
2.4 (2.5)
10,100 (4000)b
2.0 (0.0)
376 (147)
3.8 (2.6)
535 (119)
51 (35)
2670 (1030)
6.5 (4.1)c
722 (360)c
1.3 (0.5)
2460 (565)
2.5 (2.4)
1600 (2400)
1.3 3 106
(6.2 3 104)b
216 (38)b
7.6 3 105
(4.1 3 105)b
173 (84)b
1.9 3 103
(0.6 3 103)
4.9 (0.4)
3.7 3 103
(1.9 3 103)
3.5 (2.0)
3.2 3 105
(1.2 3 104)
112 (17)
8.8 3 103
(2.8 3 103)c
5.6 (3.5)
1.2 3 104
(0.5 3 104)
17.8 (8.5)
1.3 3 104
(0.6 3 104)
3.9 (1.2)
Note. Underlined values designate males and females statistically different, p 0.05.
a
Tmax (females), Cmax, AUCinf, and t1/2 statistically different from 8-2 FTOH, p 0.05.
b
Cmax, AUCinf, and t1/2 statistically different from 8-2 FTOH, p 0.05.
c
Tmax, Cmax, and AUCinf were calculated to be 91.5, 963, and 4.0 3 104, respectively, if suspect plasma concentration values after 48 h were included.
Metabolite Identification
A comprehensive list of metabolites identified in urine, bile,
feces, and plasma following a single oral dose of 8-2 FTOH to
rats, along with observed fragment ions, is presented in Table 5.
TABLE 4
Material Balance at 168 h following a 125 mg/kg Single 6-h
Dermal Exposure to [3-14C] 8-2 FTOH to Male and
Female Rats (n ¼ 3 rats per sex)
Percentage of
14
C administered dose
Males
Urine
Feces
Skin wash
Body wrap
Cage wash
Charcoal trap
Residual feed
Tissuesa
Total recovered
Total absorbeda,b
Females
Mean
SD
Mean
SD
0.04
0.18
34.1
0.22
0.01
37.7
0.01
0.79
73.1
1.03
0.01
0.06
3.47
0.07
0.01
6.44
NA
0.09
5.83
0.06
0.05
0.10
29.3
0.17
0.02
38.3
0.03
0.28
68.2
0.46
0.01
0.06
4.36
0.02
0.01
13.3
NA
0.06
17.7
0.15
Note. NA ¼ SD not determined as radioactivity was only detectable in less
than or equal to two samples.
a
Males and females statistically different, p 0.05.
b
Sum of urine, feces, cage wash, residual feed, and tissues.
Urine metabolites. LC-ARC analysis of urine following
oral dosing showed an initial peak, identified by mass spectral
analysis as PFO, which eluted near the solvent front (Fig. 2a).
Elution of PFO with the solvent front likely resulted from
channeling within the FluroFlash column due to the large
injection volume used to detect the low-concentration metabolites. Column-retained PFO eluted at approximately 31 min.
Of the urinary metabolites resolved via fraction collection and
mass spectral analysis, PFO concentrations, which included
unretained PFO, were highest (~ 25% of urinary metabolites),
accounting for approximately 0.48 and 1.16% of the administered 14C dose, at the high- and low-dose levels, respectively.
The second LC-ARC peak (Unknown-1) daughter ion mass
spectrum (m/z 429) contained two fragment ions, m/z 253 and
m/z 176; the loss of 176 amu is diagnostic for glucuronide
conjugates. However, given the proposed mass, this conjugate
of undetermined structure would not possess the radiolabel
so the actual radiolabeled metabolite associated with this
LC-ARC response is not known. Coeluting perfluorohexanoate
(PFHx) and 5-2 FTOH (Unknown-2) and 7-2 (secondary)
FTOH glucuronide (7-2 sFTOHGluc) were identified in
fractions from the pooled sample and accounted for < 0.5%
of the administered dose. Since neither the six-carbon PFHx
nor the 5-2 FTOH would contain the 14C radiolabel (position 3
of 8-2 FTOH), the LC-ARC response was likely due to an
unstable metabolite not detected by LC-MS (Fig. 2a). Reanalysis of the urine pool 2 months subsequent to the initial
evaluation and following a number of freeze-thaw cycles
confirmed the loss of two peaks, one associated with the
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
347
TABLE 5
Metabolites Observed in Urine, Feces, Bile, and Plasma following a Single Oral Dose of
[3-14C] 8-2 FTOH to Male and Female Rats
Molecular weight (amu)
314
364
414
430
440
442
458
464
464
472
478
545
559
587
590
601
602
616
640
731
745
Product ions (m/z)
119, 269
119, 169, 319
169, 219, 369
113, 253
169, 219, 355, 369, 395
317, 337
393
419
165, 241, 253, 403, 423,
463 (M-H), 523 (MþAcO)
Weak signal
393
Weak signal
471
Weak signal
75, 113, 175, 330, 373
Weak signal
280, 485, 581
471
89, 131, 193, 619
254, 272, 306, 417, 457
254, 272, 471
Identification
PFHx
PFHp
PFO
Unknown glucuronide
7-3 uAcid
7-3 Acid
8-2 FTUA
PFN
8-2 FTOH
8-2
8-2
8-2
8-2
8-2
7-2
8-2
8-2
8-2
8-2
8-2
8-2
FTUA 3-thiol
FTA
uFTOH cysteine conjugate
FTUA cysteine conjugate
uFTOH N-acetyl cysteine conjugate
sFTOH glucuronide
FTUA N-acetyl cysteine conjugate
uFTOH cysteinylglycine conjugate
FTUA cysteinylglycine conjugate
FTOH glucuronide conjugate
uFTOH GSH conjugate
FTUA GSH conjugate
isolated fraction containing PFHx and 5-2 FTOH and the other
the last unidentified peak in the initial LC-ARC profile. This
confirmed that at least two metabolites observed in the initial
LC-ARC profile were not detected in subsequent analysis either
because they had volatilized or degraded or they could not be
detected by LC-MS. Additional and preliminary follow-up experiments with urine incubated with 2,4-dinitrophenylhydrazine,
with GC-MS analysis not previously attempted in any experiments, revealed the presence of a fluorotelomer aldehyde
tentatively identified as 7-3 unsaturated FTAL (7-3 uFTAL),
which could account for the loss of at least one of the original
metabolites observed in urine. The 5-2 FTOH was probably
a contaminant in the starting technical or radiolabeled 8-2
FTOH source materials. However, the 7-carbon 5-2 FTOH was
not detected in the neat starting materials by GC-MS characterization, and so its source remains unresolved. The 7-2
sFTOHGluc, a minor metabolite in these studies (< 0.5% of
the administered dose), has been previously reported as
a primary metabolite in activated sludge (Wang et al., 2005a).
Other minor metabolites identified by the LC-ARC chromatogram (< 0.1% of the administered dose) were not resolved by
LC fraction collection and subsequent LC-MS identification.
Mass spectral analysis of a neat, pooled urine sample, using
a Zorbax C18 column with a similar mobile-phase composition
as was used with the FluroFlash column, demonstrated the
presence of similar metabolites as was observed with LC-ARC
(Fig. 2b). An MS-only screening process allowed for identification of metabolites lacking the radiolabel (position 3 on the
Urine
Feces
O
O
O
O
O
Bile
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Plasma
O
O
O
parent molecule), allowing for a more comprehensive evaluation of the metabolism of 8-2 FTOH.
Bile metabolites. The LC-ARC profiles of pooled bile from
either oral dose level revealed the presence of three principal,
late-eluting peaks at 35–40 min, independent of sex. LC-MS
analysis of isolated peaks of interest was unsuccessful.
However, LC-MS analysis of a representative neat bile sample
(Fig. 3), which was qualitatively similar to the LC-ARC
profiles, identified the metabolites m/z 744, 730, and 639,
which were proposed to be the GSH conjugate of the 8-2
FTUA, the GSH conjugate of the 8-2 uFTOH, and the
glucuronide conjugate of the parent 8-2 FTOH, respectively.
The negative electrospray ionization MS responses for these
metabolites were too weak to obtain daughter ion mass spectra.
However, the mass spectra for these metabolites compared with
those confirmed by MS-MS from the rat hepatocyte experiment
(Fig. 4) provide confidence in the structure assignment for
these three biliary metabolites (Fig. 5).
Based on the total radioactivity eliminated via the bile, these
three conjugates represented approximately 42 and 20% of the
administered 14C dose or approximately 79 and 74% of the absorbed dose for the low and high oral dose groups, respectively.
Fecal metabolites. The LC-ARC profiles of extracts from
pooled feces from either oral dose level revealed the presence
of a number of late-eluting peaks, dominated by a single,
highly organic-soluble peak. Radiochromatograms were comparable between males and females and were independent of
348
FASANO ET AL.
FIG. 2. Analysis of urine (a 24-h pooled sample) collected from a rat receiving a single oral dose of [3-14C] 8-2 FTOH. Panel a: representative LC-ARC
radiochromatogram. Panel b: LC-MS–negative single-ion chromatogram (overlay).
dose level, suggesting similar metabolite profiles. The most
predominant component, identified as 8-2 FTOH by LC-MS,
represented the greatest percentage of the administered dose
(20–38%) and a significant portion of the radioactivity burden
in the feces (51–80%). The more polar minor metabolites
(Fig. 6) were tentatively identified as the cysteine (m/z 558) and
cysteinylglycine conjugates (m/z 615) of 8-2 FTUA and the
cysteine and cysteinylglycine conjugate of 8-2 uFTOH, m/z
544 and m/z 601, respectively.
Plasma metabolites. Overlaid LC-MS–negative single-ion
chromatograms for a representative plasma sample are presented in Figure 7. Although the signal strength for the earliest
eluting component (m/z 313) was too weak for MS-MS
analysis, in-source fragmentation during full-scan analysis
indicated the loss of CO2, consistent with the structure of
PFHx. Perfluoroheptanoate (PFHp), an odd-numbered perfluorinated acid, was also observed (m/z 363) in plasma. The
most abundant plasma metabolite, assuming equivalent MS
response, was PFO (m/z 413). Numerous low-concentration
metabolites (m/z 445, 463, 439, 457, and 477) were also detected in the plasma, and the trace peak, m/z 463, was proposed
to be the odd-numbered PFN. Although the mass spectral
response for PFN was too low for structure confirmation by
MS-MS, in-source fragmentation supports this structural
assignment (the fragment ion m/z 420 is consistent with eight
perfluorinated carbons). The final metabolite, m/z 441, was the
second most abundant metabolite in plasma and possibly the
principal metabolite accounting for the majority of the
unquantified plasma metabolite burden. The daughter ion mass
spectrum of this metabolite was consistent with the structure
for the metabolite 7-3 Acid, showing responses at m/z 337 and
317 indicating successive losses of hydrogen fluoride (HF).
DISCUSSION
[3-14C] 8-2 FTOH Recovery and Distribution
In oral dosing pilot experiments with [3-14C] 8-2 FTOH in
the rat at 125 mg/kg using a closed-system environment,
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
349
FIG. 3. LC-MS–negative single-ion chromatogram (overlay) of metabolites detected in bile following a single oral dose of [3-14C] 8-2 FTOH in rats.
approximately 0.3% of the administered radioactive dose was
eliminated as organic volatiles, with barely detectable concentrations of 14CO2 (< 0.1%). Based on these initial results,
collection of 14C-organic volatiles and 14CO2 was excluded
from the main study experiments, consistent with standard
ADME practice (USEPA, 1998). Following oral administration
of radiolabeled 8-2 FTOH to male and female rats in the main
study experiments, 34–50% of the parent 8-2 FTOH transited
the gastrointestinal (GI) tract unmetabolized and was eliminated in the feces. It is plausible that the actual amount of
unmetabolized parent may have been higher, particularly at the
125-mg/kg dose level, since radiolabeled 8-2 FTOH was shown
to have been lost from the feces during sample processing due
to its volatility and low water solubility.
Only a small portion of the total radioactivity in the
administered oral dose was retained in the tissues after 7 days
following a 5-mg/kg (7%) or 125-mg/kg (4.4%) single oral 8-2
FTOH dose. The percentage of dose associated with the fat at
168 h, based on a body weight of 200 g, and USEPA’s (1994)
relationship for total body fat as a function of body weight, was
estimated to be 1.2–1.4% (male) and 2.1–2.8% (female) at the
low- and high-dose levels, respectively. In pilot experiments,
attempts were made to resolve the character of the radioactivity
in fat, which contained the highest radioactivity residues at
168 h postdose. However, LC-ARC analysis of an ethanolwater (vol/vol) extract from a representative sample proved
unsuccessful. An additional evaluation of the extract by
LC-MS revealed the presence of short-chain perfluorinated
FIG. 4. LC-MS–negative single-ion chromatogram (overlay) of metabolites detected in extract sample from rat hepatocytes incubated in vitro for 120 min at
37C with 8-2 FTOH.
350
FASANO ET AL.
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
351
FIG. 6. LC-MS–negative single-ion chromatogram (overlay) of metabolites detected in feces following a single oral dose of [3-14C] 8-2 FTOH in rats.
acids (PFHx and PFHp), including PFO and PFN, independent
of sex, but these perfluorinated acids were also observed in fat
from a control sample. Clearly, more work is needed to clarify
and confirm the presence of these perfluorinated acids in
selected tissues and whether or not their presence is due to
exposure to and metabolism of 8-2 FTOH.
Dermal exposure to [3-14C] 8-2 FTOH in vivo resulted in
significant amounts of the applied dose volatilizing from the
skin surface during the exposure and being removed by
washing (68–72%). Efforts to increase the recovery of the
applied [3-14C] 8-2 FTOH dose were improved by the use of
ethanol instead of a soap and water solution to wash the skin at
the end of the 6-h exposure; pilot experiments where a 2% soap
and water solution was used resulted in unacceptable recoveries (< 40%), which increased significantly with the unconventional use of ethanol. Based on recent in vitro dermal
experiments conducted at DuPont (DuPont-6997, unpublished)
using rat and human skin mounted in a static diffusion cell
model and a 6-h exposure to [3-14C] 8-2 FTOH in an ethanol
vehicle, a conservative model for exposure to neat 8-2 FTOH
total absorption was ~ 50% and ~ 30% for rat and human skin,
respectively. In vitro dermal absorption of 8-2 FTOH from
a 0.5% methylcellulose vehicle was much lower for human skin
(~ 7%). Based on the in vitro (ethanol vehicle) and in vivo
dermal experiments, human in vivo exposure to 8-2 FTOH
would probably result in < 1% absorption with a majority of
the absorbed dose becoming sequestered within the stratum
corneum, which would eventually be lost by normal desquamation (Reddy et al., 2000; Roberts and Walters, 1998;
Schaefer and Redelmeier, 1996).
8-2 FTOH Metabolic Pathway in the Rat
A proposed metabolic pathway for 8-2 FTOH in the rat,
which consists of a conjugate route and a degradation route, is
presented in Figure 8.
The conjugate metabolic pathway involves direct conjugation of parent with glucuronic acid. Although the supporting
in vitro experiments with rat hepatocytes suggest that 8-2
FTOH may also be conjugated to sulfate, no sulfate conjugates
were identified in the rat in vivo oral dosing studies. A second
conjugate pathway involves sequential metabolism of 8-2
FTOH to 8-2 FTAL, elimination of HF to form the unsaturated aldehyde (8-2 uFTAL), conjugation of 8-2 uFTAL with
GSH, and finally reduction of the unsaturated aldehyde conjugate to 8-2 uFTOHGS. The 8-2 uFTOHGS conjugate was
apparently further degraded by enzymes of the mercapturic
acid pathway yielding the cysteinylglycine (8-2 uFTOHSCysGly) and cysteine conjugates (8-2 uFTOHSCys). The
mercapturate, N-acetylated 8-2 uFTOH cysteine conjugate
(8-2 uFTOHSCysNAcetyl), which was preferentially eliminated in urine and likely formed via 8-2 uFTOHSCys, facilitated by N-acetyltransferase, could either be converted back to
the deacetylated form via a deacetylatase (DA) or directly acted
upon by cysteine conjugate b-lyase (CCbL) to yield 8-2 uFTOH
3-thiol, although the 8-2 uFTOH 3-thiol metabolite was not
identified in the current investigation. Additionally, 8-2
FTUAGS and its degradation products (8-2 FTUASCysGly,
8-2 FTUASCys, and 8-2 FTUASCysNAcetyl) were detected
in bile and feces, respectively. The 8-2 FTUASCysNAcetyl
FIG. 5. LC-MS–negative ion (top) and daughter ion (bottom) mass spectra of metabolites detected in bile following a single oral dose of [3-14C] 8-2 FTOH in
rats: m/z 744 (panel a), 730 (panel b), and 639 (panel c).
352
FASANO ET AL.
FIG. 7. LC-MS–negative single-ion chromatogram (overlay) of a plasma pool (0.5–24 h) from a male rat following a single 5-mg/kg oral dose of [3-14C] 8-2
FTOH (x ¼ unresolved metabolites: m/z 445, 463, 439, 457, and 477).
could be deacetylated and metabolized via CCbL to the 8-2
FTUA 3-thiol, which was observed in plasma.
The first steps in the degradation metabolic pathway are
oxidation of 8-2 FTOH to 8-2 FTA by the sequential action of
alcohol dehydrogenase or P450 and aldehyde dehydrogenase,
with 8-2 FTAL as a transient intermediate. 8-2 FTUA was
formed by dehydrofluorination of 8-2 FTA and is likely a key
branching point in the degradation metabolic pathway. In the
first branch, 8-2 FTUA undergoes reduction and dehydrofluorination yielding 7-3 uAcid, which was identified in plasma; 7-3
Acid can be formed by reduction of 7-3 uAcid and was a stable
metabolite observed in these studies and previous microbial
degradation experiments (Wang et al., 2005b). Both the
saturated and unsaturated 7-3 Acids may enter the mitochondrial fatty acid b-oxidation pathway generating PFO. A second
degradation branch is proposed to lead to the formation of 7-2
sFTOH, a primary metabolite identified in microbial cultures
from either 8-2 FTUA or 7-3 uAcid, via the intermediates 8-1
Olefin and 7-2 Olefin (Wang et al., 2005b). A glucuronide
conjugate of 7-2 sFTOH (7-2 sFTOHGluc) was observed in
urine from rats in the current in vivo oral dosing experiment and
supports the formation of the secondary alcohol precursor.
Most interesting is the observation of several perfluorocarboxylates with less than eight carbons but no shorter than five
carbons. These metabolites indicate mammalian metabolism
pathways that resulted in multiple CF2 groups transforming, a
finding not previously reported. The formation of trace amounts
of odd-numbered perfluorocarboxylates has been reported to
occur in activated wastewater sludge following incubation with
8-2 FTOH (Pace Project CA085, 2002). Formation of PFN from
8-2 FTA could occur via a-oxidation; a-oxidation in mammals
has been previously reported for phytanic acid, a branchedchain fatty acid (Casteels et al., 2003; Wanders et al., 2003), and
2-hydroxyoctadecanoic acid, a straight-chain 2-hydroxyfatty
acid (Foulon et al., 2005). Nonetheless, the metabolic pathways
involved in the formation of the even-numbered (PFHx, PFO)
and odd-numbered perfluorocarboxylates (perfluoropentanoate,
which was observed in rat hepatocytes, and PFHp and PFN)
warrant additional investigation.
The Relationship between 8-2 FTOH Metabolism and
Toxicity in the Rat following Subchronic Exposure
In the rat, metabolism of 8-2 FTOH involved dehydrofluorination. These dehydrofluorination metabolic steps likely
resulted in excess concentrations of fluoride in plasma at 125
mg/kg/day (~ 200–460 ng/ml) compared to controls (~ 100 ng/
ml) following subchronic exposure (DuPont-9478, unpublished) and caused the toxicologically significant degenerationdisorganization of enamel organ ameloblasts cells; high urinary
fluoride concentrations were also noted in the 90-day study.
The tooth lesion, along with the increased incidence of striated
teeth, was concluded to be consistent with fluoride toxicosis
(Reddy and Hayes, 1989).
Since only a minor portion of the parent FTOH was converted
to perfluorinated acids and a majority was metabolized to 8-2
FTUAGS and the 8-2 uFTALGS with subsequent reduction to
8-2 uFTOHGS, depletion of hepatic and extrahepatic GSH
stores, precipitating an accumulation of 8-2 uFTAL and 8-2
FTUA, may possibly account for some of the lesions observed in
the liver following repeated exposures to 8-2 FTOH and FTOH
mixtures (DuPont-9478, unpublished; Ladics et al., 2005). In
light of a lack of supporting adverse changes in clinical
chemistry parameters, the mechanisms for the renal tubule
lesions observed following subchronic exposure to 8-2 FTOH
remain speculative (DuPont-9478, unpublished). However, it
ABSORPTION, DISTRIBUTION, METABOLISM, AND ELIMINATION OF 8-2 FLUOROTELOMER ALCOHOL IN THE RAT
353
FIG. 8. Proposed metabolic pathway for 8-2 FTOH in the rat. Conjugate pathway: UDP-glucuronosyltransferase (UDPGT); sulfotransferase (SULT);
glutathione-S-transferase (GST); deacetylase (DA); N-acetylase (NAT); cysteine conjugate b-lyase (CCbL). Degradation pathway: alcohol dehydrogenase (ADH);
aldehyde dehydrogenase (ALDH).
seems plausible that the reported cortical tubule hypertrophy
may have been initiated by 8-2 uFTOH 3-thiol and/or 8-2 FTUA
3-thiol, the respective degradation products of the 8-2
uFTOHSCys and 8-2 FTUASCys, mediated by CCbL, which
is a well-described pathway for production of nephrotoxic thiol
and thioketene metabolites from GSH conjugates (Elfarra and
Anders, 1984; Green et al., 2003; Lash et al., 1986, 1998, 2002).
Recently, Kudo et al. (2005) reported a dose-dependent
increase in liver weights accompanied by peroxisome proliferation in mice administered 8-2 FTOH in diet. The observed
gross pathology and biochemical changes correlated with PFO
(not PFN) concentrations in the liver. Induction of peroxisomal
b-oxidation by PFCAs is well documented (Borges et al., 1992;
Harrison et al., 1988; Kawashima et al., 1995; Kudo et al.,
2000). Even though liver PFO concentrations were not determined in the current 8-2 FTOH ADME experiments, the
plasma kinetic results did suggest that PFO clearance was
delayed in male rats at the 125-mg/kg dose level and thus likely
to be present in the liver. Kemper and Jepson (2003) demonstrated that PFO concentrations were generally higher in liver
compared to plasma following oral administration of ammonium perfluorooctanoic acid. These observations, along with
the hepatic peroxisome proliferation observed at 125 mg/kg/day
in male rats following a 90-day dosing period and a 3-month
recovery phase (DuPont-9478, unpublished), suggest that PFO
may be partly responsible for the observed hepatic toxicity.
In summary, the studies and results described here provide
a more comprehensive picture of 8-2 FTOH ADME in the rat
354
FASANO ET AL.
than has been previously reported. Additional studies will be
required to refine the biotransformation pathways for 8-2
FTOH and to clarify the link between 8-2 FTOH metabolism
and toxicity in the rat.
ACKNOWLEDGMENTS
E. I. du Pont de Nemours & Co., Inc. (DuPont) sponsored the 8-2 FTOH
definitive experiments. Preliminary (pilot) experiments were sanctioned and
subsidized by the Telomer Research Program, whose member companies
included AGA Chemicals (Asahi Glass), Clariant GmbH, Daikin America,
Inc., and DuPont. The authors thank Dr. Ning Wang for his helpful discussions
and comments regarding the 8-2 FTOH metabolic pathway.
Krusic, P. J., Marchione, A. A., Davidson, F., Kaiser, M. A., Kao, C. P. C.,
Richardson, R. E., Botelho, M., Waterland, R. L., and Buck, R. C. (2005).
Vapor pressure and intramolecular hydrogen bonding in fluorotelomer
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Kudo, N., Bandai, N., Suzuki, E., Katakura, M., and Kawashima, Y. (2000).
Induction by perfluorinated fatty acids with different carbon chain length of
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119–132.
Kudo, N., Iwase, Y., Okayachi, H., Yamakawa, Y., and Kawashima, Y. (2005).
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