DEQ-CFW_00000137

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ELSEVIER Toxicology and Applied Pharmacology 198 (2004) 231-241
www.elsevier.com/locate/ytaap
Review
The developmental toxicity of perfluoroalkyl acids and their derivatives
Christopher Lau, a,* John L. Butenhoff,b and John M. Rogersa
"Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development,
U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
63M Medical Department, St. Paul, MN 55144, USA
Received 20 August 2003; accepted I 1 November 2003
Available online 8 April 2004
Abstract
Perfluoroalkyl acids such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have applications in numerous
industrial and consumer products. Although the toxicology of some of these compounds has been investigated in the past, the widespread
prevalence of PFOS and PFOA in humans, as demonstrated in recent bio-monitoring studies, has drawn considerable interest from the
public and regulatory agencies as well as renewed efforts to better understand the hazards that may be inherent in these compounds. This
review provides a brief overview of the perfluoroalkyl chemicals and a summary of the available information on the developmental
toxicity of the eight -carbon compounds, PFOS and PFDA. Although the teratological potentials of some of these chemicals had been
studied in the past and the findings were generally unremarkable, results from recent postnatal studies on developmental and reproductive
indices have prompted consideration of their relevance to human health risk. Based on current understanding of the developmental effects
of PFOS and PFOA in rodents, several avenues of research are suggested that would further support the risk assessment of these
perfluorinated organic chemicals.
© 2004 Elsevier Inc. All rights reserved.
Keywords: PFOS; PFOA; PFBS; PFDA; Developmental toxicity; Rodents; Review
Environmental exposures of perfluoroalkyl acids
In recent years, perfluoroalkyl acids have been identi-
fied at low parts per billion (ppb) concentrations in
samples of human serum taken from the general popula-
tion as well as certain wildlife samples (Giesy and
Kannan, 2001, 2002; Hansen et al., 2001- Hoff et al.,
2003a, 2003b; Kannan et al., 2001a, 2001b, 2002a,
2002b, 2002c, 2002d; Martin et al., 2004; Olsen et al.,
2002, 2003a, 2004; Sottani and Minoia, 2002). The
Abbreviations: POSF, perfluorooctane sulfonyl fluoride; PFOS, per-
fluorooctane sulfonate; PFBS, perfluorobutane sulfonate; PFOA, perfluor-
ooctanoic acid; PFDA, perfluorodecanoic acid.
'a This document has been reviewed in accordance with the U.S.
Environmental Protection Agency policy and approved for publication.
Approval does not signify that the contents reflect the views of the Agency,
nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
* Corresponding author, Reproductive Toxicology Division, National
Health and Environmental Effects Research Laboratory, Office of Research
and Development, U.S. Environmental Protection Agency, Mail Drop 67,
Research Triangle Park, NC 27711. Fax: [phone].
E-mail address: [e-mail] (C. Lau).
0041-008X/$ - see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j. taap.2003.11.031
presence of organic fluoride in humans was actually first
reported by Taves (1968) and Shen and Taves (1974)
over 30 years ago and the organic form was originally
suggested to be perfluorooctanoate (PFDA) or, possibly,
perfluorooctane sulfonate (PFOS) (Taves et al., 1976). It
is only in the past several years that significant advances
in analytical chemistry have enabled routine detection of
individual perfluoroalkyl acids in various matrices in the
ppb or lower range by high-performance liquid or gas
chromatography followed by electrospray tandem mass
spectrometry (ES/MS/MS) (Hansen et al., 2001; Hebert et
al., 2002; Martin et al., 2002; Sottani and Minoia, 2002).
Equipped with such improvement in detection sensitivity,
Olsen et al. (1999, 2001, 2003b, 2003c) reported mean
serum PFOS levels of 1-2 ppm (range 0.1-13 ppm) and
mean PFOA serum concentrations (Olsen et al., 1998,
2000, 2003b) of approximately 5 ppm (range <0.1-114
ppm) in 3M fluorochemical production workers. A broad
survey of individual blood samples from adult Red Cross
blood donors (Olsen et al., 2003a), children from a
clinical trial (Olsen et al., 2002), and elderly subjects
enrolled in a longitudinal study (Olsen et al., 2004)
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232 C. Lau et al. / Toxicology and Applied Pharmacology 198 (2004) 231-241
indicated geometric mean serum concentrations of PFOA,
perfluorohexanesulfonate (PFHS), and PFOS in these
populations of approximately 4-5, 2-5, and 30-40
ppb, respectively. More recently, Olsen et al. (2003d)
reported liver concentrations of PFOS ranging from
<4.5 to 57 ppb in human organ donors from the general
population. The hepatic levels of PFOA in most of the
organ donor subjects were below current detection limits.
Several recent studies by Giesy and Kannan (2001,
2002), Kannan et al. (2001a, 200Ib, 2002a, 2002b,
2002c, 2002d), and others (Hoff et al., 2003a, 2003b;
Martin et al., 2004) have reported detection of PFOS in a
variety of wildlife species including fresh water and
marine mammals, fish, birds, and shellfish. Although
distribution of the chemical appears to be widespread in
the northern hemisphere, including remote locations in the
Arctic and North Pacific Oceans, concentrations of PFOS
in these animals are generally greater in the more popu-
lated and industrial regions. These investigators and others
(Martin et al., 2003b) also suggested that PFOS can be
bio-magnified in the top levels of the food chain. Although
PFOA was also found occasionally in the serum and liver
of various wildlife species at different geographic loca-
tions, concentrations are generally lower than those of
PFOS (Kannan et al., 2002a, 2002b, 2002d).
The prevalence of these perfluoroalkyl acids in humans
and wildlife populations has thus drawn considerable
attention from regulatory agencies (US EPA, 2003a,
2003b) and the public (Renner, 2001). Largely because
of the observed broad distribution and persistence of PFOS
and PFOA in humans and the environment, 3M Company
(one of the chief manufacturers) phased out production of
these chemicals by the end of 2002; although other
perfluoroalkyl acid products are poised to take up the void
left by PFOS in the commercial markets. In addition, the
Center of Disease Control and Prevention (CDC) has
recently nominated PFOS and PFOA to be included in
their National Health and Nutrition Examination Survey
(NHANES) for future exposure reports, in order to provide
a better assessment of the distribution of these chemicals
among human populations. It is therefore timely to review
the toxicology of these perfluorochemicals, and this paper
will focus on the current understanding of developmental
toxicity of these compounds.
A search of the extant literature revealed only limited
peer -reviewed publications on this topic. Because the issues
related to developmental toxicity of these chemicals arise
from recently completed studies, manuscripts are still in
preparation and substantial toxicity data are available only
in the form of reports submitted to the U.S. EPA and other
regulatory agencies. Information from the latter source is
included in this review and details of these studies are
generally accessible to the public upon request from these
agencies.
As a class, these perfluorochemicals share unique phys-
ical and chemical properties that are related to their com-
mercial applications as well as potentials for toxicity. A
clear understanding of these properties will be useful for
toxicologists and risk assessors in evaluating the toxic
potentials of these compounds. Thus, although the focus
of this review is directed toward the developmental toxicity
of perfluoroalkyl acids, a brief background on their chem-
istry, environmental distribution, and pharmacokinetic pro-
files is also provided for the readers' purview.
Chemistry, production, and uses of perfluoroalkyl acids
Organic fluorochemicals are compounds in which one
or more carbon —hydrogen (C-H) bonds are replaced by
carbon —fluorine (C-F) bonds. Fluorine is one of the most
reactive elements in ionic form and one of the most stable
in bound form. Hence, the fully fluorinated hydrocarbons,
or perfluorocarbons, are stable in air even at high temper-
atures (in excess of 150 °C), nonflammable, not readily
degraded by strong acids, alkalis or oxidizing agents, and
not subject to photolysis. The unique stability of these
chemicals thus renders them practically nonbiodegradable
and thus, very persistent in the environment (Key et al.,
1997, 1998; Prescher et al., 1985).
Naturally occurring fluorinated organic chemicals are
rare in biological systems due to the high energy required
to form or break the C-F bonds, and all known biologically
produced fluorinated organics contain only one fluorine
atom (review: Key et al., 1997). However, partially or fully
fluorinated (perfluorinated) organic molecules can be syn-
thesized in the laboratory and in full-scale manufacturing
operations.
One method for production employs the Simons Elec-
trochemical Fluorination (ECF) process that was discovered
in 1937. The ECF process is based on the reaction between
organic feedstocks such as 1-octanesulfonyl fluoride
(C8H17SO2F) and anhydrous hydrogen fluoride (HF), which
is fueled by an electrical current, causing the hydrogen
atoms on the carbon backbone to be replaced by fluorine
atoms, with resultant products such as perfluorooctane
sulfonyl fluoride (POSF, C$F17SO2F). However, during this
process, fragmentation and rearrangement of the carbon
skeleton can occur, producing fluorinated organic molecules
of various carbon chain lengths (less than eight) and a
mixture of linear, branched, and cyclic isomers of the
chemical. Typically 70-85% of the mixture was linear
and 15-301X0 was a mixture of branched isomers. 3M has
produced fluorochemicals by the ECF process for industrial
and consumer use since the 1950s, although this manufac-
turer has now phased out essentially all production of
perfluorooctanyl (PFOA and POSF-based) chemistries.
An alternative production method for the functionalized
perfluoroalkyl acids involves telomerization of tetrafluoro-
ethylene units, and many manufacturers employ this tech-
nology to produce materials that, in some cases, have
similar properties to the ECF-based materials. In this pro-
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C. Lau et al. / Toxicology and Applied pharmacology 198 (2004) 231-241
cess, a telogen such as pentafluoroethyl iodide (F(CF2CF2)I)
polymerizes with terafluoro ethylene (CF2CF2) to form
telomer intermediates of desired carbon chain length (typ-
ically up to 12); the resultant telomer iodides are then
reacted with ethylene via free radical addition. The telome-
rization process thus yields straight -chain alcohols
(F(CF2CF2) CH2CH2OH) that can be converted into final
products for commercial application. In general, these
telomer-derived products may consist of a distribution of
various carbon chain lengths. Hence, the fluorinated organic
chemicals found in the environment represent a family of
compounds of various sizes and isomeric forms.
The high ionization potential and low polarizability of
fluorine lead to weak inter- and intramolecular interactions
that are reflected by the extremely low surface tension of the
perfluoroalkyl acids. The partitioning behavior of perfluor-
oalkyl acids is also unique; when they are mixed with water
and hydrocarbons, three immiscible phases are formed,
indicating the hydrophobic and oleophobic nature of these
compounds. By attaching a charged moiety such as carbox-
ylic acid, sulfonic acid, or phosphate to the perfluorinated
chain, the molecule becomes more water soluble due to the
hydrophilic nature of the added functional group. Together,
the physical properties of the functionalized perfluoroalkyl
acids render these compounds ideal surfactants (Kassa,
2001). Although all amphoteric perfluoroalkyl acids share
some surfactant properties, the eight -carbon chemicals such
as POSF-based materials, PFOS and PFOA have been most
effective in commercial uses. Indeed, POSF- and telomer-
related products are found in over 200 known industrial and
consumer applications ranging from water-, soil-, and stain -
resistant coatings for clothing fabrics, leather, upholstery,
and carpets, to oil -resistant coatings for paper products
approved for food contact, electroplating, and electronic
etching bath surfactants, photographic emulsifier, aviation
hydraulic fluids, fire -fighting foams, floor polishes, and
insecticide formulations (Renner, 2001; Seacat et al.,
2002). According to 3M, the global production of POSF
for 2000 (the last year of active production) was estimated at
about 3,545 metric tons, and in 2001, the worldwide
production was 175 metric tons (3M, 2003). Information
on PFOA production by various manufacturers is not readily
available, but can be estimated in excess of 500 metric tons
per year.
In commercial applications, POSF was converted into
other intermediates such as acids, alcohols, and sulfona-
mides for further polymerization and csterification, but
some of these compounds and their intermediates have the
potential to break down metabolically or in the environment
to PFOS as an end -stage metabolite and product. PFOA
(primarily ammonium salt) can be used as a surfactant and
an emulsifier in the production of polytetrafluoroethylene as
well as other fluoropolymers and fluoroelastomers.
Telomer-based molecules may have the potential to degrade
metabolically to perfluorinated acids (Hagen et al., 1981).
Most of the polymers based on fluorochemistry tend to be
233
relatively stable and resistant to degradation. Current under-
standing of fluorochemistry suggests that PFOS and PFOA
are substantially different compounds, and that toxicological
evaluation of these two chemicals should be considered
separately.
Distribution of perfluoroalkyl acids in the environment
POSF is a volatile liquid at normal temperature and
pressure; however, PFOS and PFOA have very low volatil-
ity and vapor pressure. Certain sulfonamide intermediary
compounds derived from POSF, such as alkyl amides, alkyl
alcohols, and alkyl acids, may sublime (3M, 2003; Schal,
1992). However, little is known about the transport and fate
of these fluorochemicals in the environment. Recent studies
reported <0.017 to 2,260 µg/1 for perfluoroalkane sulfonate
(C-6—C-8) and <0.009 to 11.3 µg/1 for PFOA in the surface
water of a Canadian tributary after an accidental release of
fire -fighting foams (Moody et al., 2002). Both PFOS and
PFOA were detected in water samples from the Tennessee
River near a 3M fluorochemical manufacturing site (Hansen
et al., 2002). Concentrations ranging from a low of approx-
imately 17 ppt (below quantitation limit) upstream of the
facility to a high of 144 ppt downstream of the site for
PFOS, and a low of 25 ppt to a high of approximately 600
ppt for PFOA were reported. Monitoring of drinking water
sources near a production plant in West Virginia indicated
the presence of PFOA at below 3 µg/l, substantially below
the interim risk screening of 150 µg/1 established by the
State and US EPA region (West Virginia Department of
Environmental Protection, 2002). Martin et al. (2002) also
reported airborne levels of certain perfluorooetanesulfona-
mide-based chemistries and telomer alcohols ranging from
approximately 10 to 400 pg/m3 in Ontario (Toronto and
Long Point), Canada.
Pharmacokinetics of PFOS and PFOA
PFOS and PFOA have been observed in retired fluro-
chemical workers to have mean elimination half-lives of
several years (Burris et al., 2002). PFOS is readily absorbed
and distributed primarily in the serum and liver (most likely
through enterohepatic circulation), but poorly eliminated
(Burris et al., 2002; Johnson and Ober, 1979; Johnson et
al., 1979, 1984; Nokcr and Gorman, 2003a; Seacat et al.,
2002). The elimination half-life of PFOS in rats and
monkeys has been estimated at >90 days for male rats
(Johnson et al., 1979) and approximately 100--200 days
for male and female cynomolgus monkeys (Noker and
Gorman, 2003a; Seacat et al., 2002). PFOA is also readily
absorbed (Kemper and Jepson, 2003); however, notable
differences in elimination between species have been
reported (Kennedy et al., 2004; Kojo et al., 1986; Kudo
and Kawashima, 2003; US EPA, 2003a). In the rat, a major
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234 C. Lau et al. / Toxicology and Applied Pharmacology 198 (2004) 231-241
gender difference exists in the elimination of PFOA, with
half-lives estimated at 1.9-24 h for females and 4.4-9 days
for males (Hanhijarvi et al., 1982; Kemper and Jepson,
2003; Kudo et al., 2002; Ophaug and Singer, 1980; 1987;
Vanden Heuvel et al., 1991a; Ylinen et al., 1990). The
gender difference in rats appears to be under steroid hor-
monal control (Kawashima et al., 1991; Kudo et al., 2002;
I-lanhijarvi et al., 1982; Vanden Heuvel et al., 1991a, 1992;
Ylinen et al., 1989). Gibson and Johnson (1983) reported
that hormonal changes during pregnancy did not appear to
alter the rate of PFOA elimination. A smaller sex -related
difference in PFOA elimination was found in beagle dogs
(Hanhijarvi et al., 1988), with plasma half-lives of 202 and
473 h estimated for females and males, respectively. How-
ever, it is noteworthy that striking gender differences in
PFOA elimination have not been observed in primates and
humans (Burris et al., 2002; Noker and Gorman, 2003b),
and appear to be unlikely in the mouse (Sohlenius et al.,
1992; Uy-Yu et al., 1990; US EPA, 2003a),
Toxicity of perfluorooctane sulfonate
The potential toxicities of PFOS in monkeys (Seacat et
al., 2002), rats (Austin et al., 2003; Seacat et al., 2003;
Thibodeaux et al., 2003), fish (Hoff et al., 2003b: Martin
et al., 2003a, 2003b), and humans (Olsen et al., 1999,
2003b, 2003c) have been characterized and summarized in
recent reviews (3M, 2003; US EPA, 2003b; link to OECD:
http://www.oecd.org/dataoeedi23/18/2382880.pclo. In brief,
subchronic exposure to PFOS led to significant weight loss
accompanied by hepatotoxicity and reductions of serum
cholesterol and thyroid hormones. Teratological studies
have been conducted in rat, rabbit, and mouse with PFOS
(potassium and lithium salts) (Case et al., 2001b; Christian
Rat
0 5 10 15 20
Postnatal Age (days)
et al., 1999a; Gortner, 1980; Henwood et al., 1994;
Thibodeaux et al., 2003; Wetzel, 1983). The findings are
in agreement between laboratories and across species
examined, and are generally unremarkable when maternal
effects are taken into consideration. Observed developmen-
tal effects include reduction of fetal weight, cleft palate,
anasarca (edema), delayed ossification of bones (sternebrae
and phalanges), and cardiac abnormalities (ventricular
septal defects and enlargement of the right atrium). None-
theless, it should be noted that a preponderance of these
structural abnormalities was found in the highest PFOS
dose groups, where significant reductions of weight gain
and food consumption were also observed in the pregnant
dams. On the other hand, maternal malnutrition is not
likely the sole factor accounting for the induction of birth
defects. Indeed, Thibodeaux et al. (2003) have shown that
equivalent or higher incidence of malformations was seen
in the mouse, compared to the rat; yet, the deficits of
maternal weight gain and food consumption in the mouse
were much less extensive than those in the rat.
When rodent dams exposed to PFOS throughout preg-
nancy were allowed to give birth, dose -dependent deleteri-
ous effects were seen in the newborns (Lau et al., 2003; Fig.
). As a reference to body burdens of the chemical, serum
levels of PFOS in the pregnant dams at term and in the
newborns are summarized in Table 1. All rat pups were born
alive, pink, and active. However, in the highest dose (10 mg/
kg) group evaluated, the neonates became pale, inactive,
and moribund within 30-60 min, and all died soon after-
ward. In the next highest dose group (5 mg/kg), the neonates
also became moribund but survived for a longer period of
time (8-12 h). Over 95% of these animals did not survive
the first day of postnatal life, and only a few pups reached
puberty. Survival improved with lower PFOS exposure, and
was about 50% at 3 mg/kg. The morbidity and mortality of
Mouse
0 5 10 15 20 25
Postnatal Age (days)
-a-Control -0-1 mg/kg t2 mg/kg -v-3 mg/kg
--F-5 mg/kg -a-10 mg/kg -*-15 mg/kg -*-20 mg/kg
Fig. 1. Effects of prenatal exposure to PFOS on postnatal survival in rats and mice. Each data point represent means ± SE of 9-18 litters for rats and 7-18
litters for mice. Results are excerpted and slightly modified from the primary study that contained the experimental details (Lau et al., 2003).
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C. Lau et at. / Toxicology and Applied Pharmacology 198 (2004) 231-241
Table 1
Concentrations of PFOS in rat maternal serum at near -term (GD 21) and in
newborn pups
Treatment dose
Maternal serum
Newbom serum
(mg/kg)
(pPm)
(pp-)
0
0.244 ± 0.044
0.188 ± 0.070
1
19.6±1.8
35.9±0.6
2
45.0±1.6
71.9±4.1
3
71.9±6.6
86.5±10.0
5
80.6±3.0
108.2±4.6
10
189.9 ± 6.6
—
Data represent means ± SE of 9-14 dams and 3-5 newborns, and are
excerpted from primary studies that contained the experimental details (Lau
et al., 2003; Thibodeaux et al., 2003).
the newborn rats appeared to be correlated to their body
burden of the fluorochemical. After the first week of
postnatal life, the mortality rate was not different between
control and treated groups. Cross -fostering the PFOS-ex-
posed rat pups (5 mg/kg) to control nursing dams immedi-
ately after birth failed to improve survival of the neonates,
thus ruling out a role for abnormal maternal behaviors
associated with the fluorochemical treatment. The maternal
dose corresponding to the BMDL5 (lower limit of the 95%
confidence interval on the Benchmark Dose that would
predict a 5% increase in response above background inci-
dence) for survival of rat pups at postnatal day 8 was
estimated at 0.58 mg/kg.
Postnatal growth of surviving rat pups was somewhat
stunted by in utero exposure to PFOS. Body weights of pups
in the 2 mg/kg and higher dose groups significantly lagged
behind those in controls, and this effect persisted past wean-
ing. Development of the PFOS-exposed pups was also
significantly hindered as the average age at eye opening
was delayed by a day, although no significant changes in
the onset of puberty were noted. Hypothyroxinemia was
observed in PFOS-exposed neonates. Because thyroid hor-
mones are known to regulate brain development, the ontog-
eny of neurochemical and neurobehavioral markers was
evaluated. Prenatal exposure to PFOS produced only mar-
ginal, but statistically significant deficits in the developmen-
tal patterns of choline acetyltransferase activity (an enzyme
marker sensitive to thyroid hormone status), and did not
affect learning and memory behaviors determined by T-maze
delayed alternation. This lack of notable changes in the CNS
in these studies may have been related to the extent of thyroid
hormone imbalance (reductions of thyroxine but not triiodo-
thyronine or TSII) induced by the prenatal PFOS treatment.
A similar profile of postnatal mortality was observed in
the mouse (Fig. 1), although the maternal dosage required
for the effect was higher than that in the rat. Most mouse
offspring of dams exposed to 15 or 20 mg/kg PFOS did not
survive for 24 h after birth. The LD50 was estimated at 10
mg/kg (compared to approximately 3 ing/kg in the rat),
whereas survival of the lower dose groups (1 and 5 mg/kg)
was not different from that of controls. The maternal dose
corresponding to the BMDL5 for survival of mouse pups at
235
postnatal day 6 was estimated at 3.88 mg/kg, approximately
six times higher than that of the rat. Among survivors, a
trend toward growth deficit was evident. Compared to the
rat, a more pronounced increase of liver weight was noted in
PFOS-exposed neonatal mice. Hepatic enlargement per-
sisted through the duration of the study (to PD 35). Serum
thyroxine levels in the developing mice exposed to PFOS
prenatally tended to be lower, but less consistently so than in
PFOS-exposed rat pups. A significant delay in eye opening
was also detected in treated mouse neonates.
Similar effects of PFOS on newborn rodents can be
achieved by different treatment paradigms. Butenhoff et al.
(2002b) treated female rats with PFOS for 42 days before
mating and throughout pregnancy at lower doses (0.4-2
mg/kg/day) and reported marked reduction of viability in
pups in the two highest dose groups (49% and 17%,
respectively, for the 1.6 and 2 mg/kg groups), as well as
significant retardation in postnatal growth among the
survivors, even in the lower dose groups. These results
confirmed those from a two -generation reproductive study
with the same treatment period for parental rats at doses of
0.1, 0.4, 1.6, and 3.2 mg/kg/day PFOS. In this study, the
1.6 and 3.2 mg/kg doses produced 34% and 100% Ft pup
mortality within the first days postpartum, respectively,
transient reductions in weight gain in F2 pups during the
lactation period at maternal doses of 0.4 mg/kg/day, and no
effects at 0.1 mg/kg/day (Christian et al., 1999a). In a
cross -foster study with the same treatment paradigm,
postpartum transfer of the PFOS-exposed pups (1.6 mg/
kg) to control nursing dams failed to reduce mortality
(Case et al., 2001a). Grasty et al. (2003b) examined critical
windows of PFOS toxicity and reported a similar increase
of neonatal mortality in the rat with exposure of pregnant
dams to high doses of PFOS (25 mg/kg/day) for a 4-day
period during various stages of pregnancy. Neonatal mor-
tality occurred after dosing in all time periods, but the
incidence of neonatal death increased as the exposure
period fell later during gestation, reaching 100% in the
treatment group of GD 17-20. In another experiment,
dosing with 50 mg/kg/day of PFOS on GD 19 and 20
only was sufficient to produce almost 100% mortality.
Hence, the neonatal mortality resulting from PFOS admin-
istration to pregnant rats does not require PFOS exposure
before day 19 of gestation, suggesting that the critical
period is late -gestational or perinatal. Interestingly, al-
though the incidence of neonatal mortality was correspond-
ingly lower in the early exposure groups, body weights of
the newborns in these groups (4-day treatment on GD2-5,
6-9, or 10-14) were significantly lower than those of
controls or late exposure groups Grasty et al. (2003b).
The pathophysiology underlying PFOS-induced neonatal
mortality is largely unknown at present. Results from Grasty
et al. (2003b) suggest that organ systems developing late in
gestation may be targets for PFOS insult. This hypothesis is
also consistent with the relatively unremarkable teratologi-
cal findings. Considering that PFOS-induced organ toxicity
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236 C. Lau et al. / Toxicology and Applied pharmacology 198 (2004) 231-241
is incompatible with postnatal survival, maturation of the
lung and pulmonary function is a plausible target.
Numerous chemicals have previously been shown to
interfere with fetal lung development (Lau and Kavlock,
1994). The profile of neonatal mortality induced by PFOS is
in fact reminiscent of the developmental toxicity of nitrofen,
a herbicide that perturbs fetal lung maturation, ultimately
leading to compromised cardiopulmonary function and
death in newborn rats (Lau et al., 1986, 1988, Stone and
Manson, 1981). Indeed, the most recent findings reported by
Grasty et al. (2003a, 2003b) are promising in support of this
contention. These investigators described significant differ-
ences in histological and morphometric evaluations of the
lung between control and PFOS-treated newborns that
suggest PFOS-induced inhibition of or delayed perinatal
lung maturation.
Nevertheless, the involvement of other possible mech-
anisms of PFOS toxicity such as interference with mito-
chondria) bioenergetics (Berthiaume and Wallace, 2002;
Starkov and Wallace, 2002), impedance of cell -cell com-
munication through gap junctions (Hu et al., 2002), inter-
actions with fatty -acid binding proteins (Luebker et al.,
2002a), hepatotoxicity (peroxisome proliferation) (Ikeda et
al., 1987; Sohlenius et al., 1993), and alterations of maternal
and fetal thyroid hormone economy ('Thibodeaux et al.,
2.003) warrants consideration, On the other hand, a recent
report did not provide evidence to support PFOS-induced
interference with cholesterol synthesis (through inhibition of
HMG CoA reductase activity, Haughom and Spydevold,
1992) as a potential mechanism for developmental toxicity
of the chemical because cholesterol or mevalonic acid
supplementation failed to ameliorate the adverse effects of
PFOS (Luebker et al., 2002b).
Toxicity of N-ethyl-N-(2-hydroxyethyl)-
p erf luoroo ctan esulfon am ide
N-ethyl-N-(2-hydroxyethyl)-perfluoro octanesulfonami de
(N-EtFOSE) represents a building block for POSF-related
products for commercial applications and can be degraded
metabolically and in the environment to PFOS. Case et al.
(2001b) evaluated its potential developmental effects in rats
(with doses of 1, 5, 10, and 20 mg/kg) and rabbits (with doses
of 0.1, 1, 2.5, and 3.75 mg/kg). In the rat, maternal body
weight gains were reduced in the high dose groups, where
reductions of fetal weight were also evident; but teratological
findings were unremarkable. In the rabbit, dose -dependent
reductions of maternal weight gain were seen and late -term
resorptions were increased at the highest dose. No structural
aberrations were noted in fetal examinations.
A two -generation reproductive study in rats was con-
ducted with N-EtFOSE (Christian et al., 1999b) at doses of
1, 5, 10, and 15 mg/kg/day, and the profile of developmental
toxicity was quite similar to that described with PFOS (Case
et al., 2001a, 2001b; Christian et al., 1999a; Lau et al., 2003;
Thibodeaux et al., 2003). Doses of the fluorochemical as high
as 15 mg/kg/day had no effect on estrous cycling, mating, or
fertility of the F0 generation rats, although a decrease in
numbers of implantations and viable embryos was noted.
Postnatal survival of the F1 generation pups was significantly
reduced in the 10 and 15 mg/kg dose groups, characterized by
stillbirth and mortality during the first 72 h of life; in addition,
weight gain deficits were evident among the survivors whose
reflex and physical development were slightly delayed. These
adverse effects were less pronounced in the lower dose
groups, and their reproductive performances were not ad-
versely affected. However, the number of F1 dams in the 5
mg/kg dose group that had stillborns or neonatal mortality
(F2) was slightly, but significantly increased, and the weight
gain of F2 pups was also reduced. There were no significant
adverse effects at 1 mg/kg/day.
Toxicity of perfluorooctanoic acid
The general toxicity profile of PFOA has been charac-
terized in monkeys (Butenhoff et al., 2002a; Griffith and
Long, 1980), rats (Abdellatif et al., 1991; Biegel et al.,
t 995, Cook et al_, 1992; Diaz et al., 1994; Griffith and
Long, 1980; Kennedy, 1985; Kennedy et al., 1986; Kudo et
al., 1999; IJu et al., 1996; Ohmori et al., 2003; Olson and
Andersen, 1983; Pastoor et al., 1987; Reo et al., 1994), mice
(Yang et al., 2000, 2001), rabbits (Griffith and Long, 1980;
Kennedy, 1985), fish (Martin et al,, 2003a, 2003b), and
humans (Gilliland and Mandel, 1993, 1996, Olsen et al.,
1999, 2000, 2003b), and summarized in a recent review
(Kennedy et al., 2004; Kudo and Kawashima, 2003). A
preliminary risk assessment of the developmental toxicity of
PFOA was recently conducted by the Office of Pollution
Prevention and Toxic Substances of the U.S. Environmental
Protection Agency (US EPA, 2003a). Hence, only an
abbreviated summary of the animal studies mentioned in
the risk assessment document will be provided here.
Because of the peculiar gender difference in the clearance
and excretion of PFOA in some animal species (vide supra),
the choice of animal models is critical when evaluating the
reproductive and developmental toxicity of this chemical.
Thus far, only information pertaining to the rat and the
rabbit is available. Teratological studies of PFOA have been
conducted by Gortner (1981) with rats and rabbits (Gortner,
1982), and Staples et al. (1984) with rats, but neither
laboratory reported any significant findings with adminis-
tered doses up to 100-150 mg/kg/day for rats and 50 mg/
kg/day for rabbits.
More recently, Butenhoff et al. (2004) undertook a two -
generation study of ammonium PFOA where rats were
given 1, 3, 10, or 30 mg/kg daily. In the F, generation,
observations during the lactational period were unremark-
able, with the exception of a significant lag of weight gain in
the offspring of the 30 mg/kg PFOA-treated group noted
during the first postnatal week and a statistically significant
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C. Lau el al. / Toxicology and Applied pharmacology 198 (2004) 231-241
decrease in the lactation index. A statistically significant
increase of mortality was seen in both male and female pups
at 30 mg/kg, especially in the first week after initiating
gavage dosing at weaning. Nine of the thirteen neonatal
deaths in the postweaning period involved the lightest pups
and occurred in the first few days postweaning, suggesting
that low body weight or immaturity at weaning was a likely
factor in the mortality of these pups. Among the survivors,
significantly reduced weight gain was observed in males,
reaching statistical significance in all treated groups by
termination of the study at 113 days postweaning. F,
females in the 30 mg/kg dose group had significant reduc-
tions in body weight after weaning and these deficits
persisted through the remainder of the study. Both Fl-
generation males and females, at the highest dose (30 mg/
kg/day) evaluated, experienced statistically significant
delays (by 2 and 4 days for females and males, respectively)
in reaching sexual maturity. However, no effects on mating
or fertility parameters were apparent, and findings in the F2
generation were unremarkable. Histological examination of
tissues by light microscopy did not reveal any abnormalities
in sex organs. However, hypertrophy and vacuolation of the
zona glomerulosa of the adrenals of P-generation (10 and 30
mg/kg) and Fl-generation (30 mg/kg) males were seen, and
hepatocellular hypertrophy was present in F, males at doses
of 3 mg/kg and higher.
Interpretation of these data and their extrapolation for
human health risk assessment are quite challenging because
sexually mature female rats possess an innate ability to
excrete PFDA rapidly, with an elimination half-life of 2-3
h, as opposed to days or weeks in male rats, dogs, and
monkeys, or years in humans. Preliminary results from a
pharmacokinetic study (Kemper and Jepson, 2003) further
complicate the issue. These investigators reported that
plasma kinetics in adult female rats appeared to be biphasic
at the two highest administered single oral doses of 5 and 25
mg/kg (unlike the monophasic profile in male rats), and
terminal elimination half-lives were dependent on the dose
level. Thus, the toxic manifestation of PFDA in female
pregnant rats, where steady state of the chemical is unlikely
to be reached on a daily gavage dosing regimen and
significant variations in the serum concentration of PFOA
occur between the treatment intervals, may be quite different
from those species (including humans) where the steady-
state kinetics is reached gradually.
The postweaning mortality and delays in pubertal onset
in the F, generation at 30 mg/kg (Butenhoff ct al., 2004)
reflect potential developmental effects of PFOA that may be
partially explained by reduced body weight at weaning or
weight gain thereafter. Interestingly, the fact that these
effects occurred in both genders suggests a lack of the sex
hormone -mediated differences in excretion. This contention
would be consistent with the observation that the organic
anion transporter protein (OAT2) that has been proposed to
be responsible for the high PFOA excretion rate in adult
female rats (Kudo et al., 2002) does not begin to be
237
expressed until sometime between postnatal days 35 and
40 (Buist et al., 2002). These data suggest that pharmaco-
kinetic profiles in sexually immature rats may be different
from those in the adults.
Toxicity of other perfluoroalkyl acids
Compared to the C-8 perfluorochemicals, less is known
about perfluoroalkyl acids of other carbon chain lengths.
Perfluorohexanesulfonate (PFHS) has been found in serum
samples from the general population including children, adult
Red Cross blood donors, and elderly subjects enrolled in a
prospective study of cognitive disorders (Olsen et al., 2002,
2003a, 2004), with geometric means of 0.002-0.005 µg/ml.
In the same studies, geometric mean serum concentrations for
PFOA were approximately 0.005 µg/ml, and, for PFOS, they
ranged from approximately 0.03-0.04 µg/ml. Perfluorobu-
tane sulfonate (PFBS) is known to have a much shorter half-
life than PFOS (Paul Lieder, 3M, personal communication);
in contrast, perfluorodecanoate (PFDA) appears to have a low
rate of elimination in the rat (Vanden Heuval et al., 199lb). In
this regard, it will be of interest in future pharmacokinetic and
structure -activity studies to determine to what extent the rate
of elimination for these fluorochemicals is directly related to
their carbon chain length.
Like PFDA, both perfluorononanoate (PFNA) and PFDA
are peroxisome proliferators (Harrison et al., 1988; Ikeda et
al., 1985; Kinney et al., .1989). The toxic potency of PFDA
is quite high, with an LD50 4.6-fold lower than that
estimated for PFOA (Olson and Andersen, 1983). PFDA
has also been reported to produce hepatotoxicity, anorexia,
alteration of fatty acid metabolism, reduction of circulating
thyroid hormones and androgen, bradycardia, and hypother-
mia in the rat (Bookstaff et al., 1990; George and Andersen,
1986; Gutshall et al., 1988, 1989, Langley and Pitcher,
1985; Pitcher and Langley, 1986; Pilcher et al., 1987; Singer
et al., 1990).
Both potassium PFBS and potassium PFHS have re-
cently been assessed for developmental and reproductive
effects. Maternal exposure to PFBS did not produce any
adverse effects on embryo/fetal development, and no
significant alterations were noted in a two -generation study
in rats at doses as high as 1 g/kg (Paul Lieder, 3M,
personal communication). PFHS did not produce develop-
mental or reproductive effects in rats at doses up to 10 mg/
kg in a onc-gencration reproductive and developmental
toxicity screening test (Hoberman and York, 2003). In
addition, Harris and Birnbaum (1989) reported a lack of
teratogenic effects of PFDA in the mouse.
Future research needs and directions
The industrial and consumer uses of perfluoroalkyl acids
and their derivatives have increased tremendously over the
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238 C. Lan et al. / Toxicology and Applied Pharmacology 198 (2004) 231-241
past half century, and the unique physical and chemical
properties of these materials makes substitution by other
organic materials difficult for some of these products and
applications. On the other hand, several of these fluorochem-
icals have become ubiquitous in the environment and the
presence of PFOS and PFOA in humans in particular has
drawn considerable attention in the public arena. Although
the toxicology of some of these compounds has been inves-
tigated in the past, the widespread exposure demonstrated in
recent bio-monitoring studies has renewed efforts to under-
stand the hazards that may be associated with these chem-
icals, and new information has come to light in recent studies.
This review has summarized recent findings from several
laboratories on the developmental toxicity of perfluoroalkyl
acids. Although the teratological potentials of some of these
chemicals had been studied in the past and the findings were
generally unremarkable, information from postnatal studies
on developmental and reproductive indices have been pub-
lished or submitted to the U.S. EPA public docket recently,
and these findings have prompted further consideration of
their relevance to human health risk. Confirmation and
elucidation of the pharmacokinetic and mechanistic bases
of these findings by different experimental approaches and
study designs will be important to establish the context of
these findings for human health risk assessment. This goal
should be attainable by active investigation within the scien-
tific community in the next few years.
Other research avenues can be useful in defining the
potential health risks of fluorochemicals. At present, little is
known about the environmental exposure, transport, and
fate of these compounds, particularly PFOA. In June 2003,
the U.S. Environmental Protection Agency began an En-
forceable Consent Agreement process with industry to
explore the environmental fate and transport of the PFOA,
telomer-based materials, and related compounds, and to
enhance understanding of the sources of PFOA in the
environment, as well as the pathways by which human
exposure to this chemical can occur. Information gathered
under this agreement will complement the data collected in
bio-monitoring programs and may lead to predictive models
of exposure. Because of their physical properties (being
hydrophobic, lipophobic and protein -bound; Ilan et al.,
2003), perfluoroalkyl chemicals may have unique profiles
of distribution in the body. Thus, pharmacokinetic charac-
terization of these compounds, especially during pregnancy
and early postnatal ages (before sexual maturity), will
greatly facilitate our understanding of chemical disposition
and potential cellular targets of PFOS and PFOA. In
addition, as information pertaining to body burden of these
fluorochemicals comes to light in the next few years (from
bio-monitoring surveys by the CDC, for instance), this
"internal dosimetric" can be used readily for cross -species
(from laboratory animal models, such as rodents and
monkeys to humans) as well as low -dose (from experimen-
tal doses to exposure levels) extrapolations for risk analysis.
More in-depth descriptive information will undoubtedly
provide additional clues to the modes and mechanisms of
PFOS and PFOA developmental toxicity in laboratory
animal models. Cellular and molecular mechanistic findings
will be instrumental in extrapolating the health risk potential
of these compounds for humans. For PFOA, potential
disparities between genders and species (i.e. pharmacoki-
netic parameters between rats and humans) have to be
reconciled to aid interspecies extrapolation. As other per-
fluoroalkyl acids are slowly coming onto the commercial
market to replace PFOS, the potential developmental toxic-
ity of these compounds should be investigated thoroughly.
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