Document xzpRZrZmQG9v7yY2G2YY4Bzg0

"McCrea, Deborah" <mccrea@taftlaw.com> 06/12/2009 03 :54 PM p. 1 To cc bcc Subject AP aas-3gq3 NCIC OPPT@EPA . Bnott, Robert A. <bilott@taftlaw.com> 06/12/2009 Letter To EPA Docket Center Taft I Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel : 513 .381 .2838 Fax: 513 .381 .0205 www.taftlaw .com / mccrea@taftlaw.com Internal Revenue Service Circular 230 Disclosure: As provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This message may contain information that is attorney-client privileged, attorney work product or otherwise confidential . If you are not an intended recipient, use and disclosure of this message are prohibited . If you received this transmission in error, please notify the sender by reply e-mail and delete the message and any attachments . From: canoncopy20a@taftlaw .com [mailto :canoncopy20a@taftlaw.com] Sent: Friday, June 12, 2009 3 :56 PM To: McCrea, Deborah Subject : 06/12/2009 Letter To EPA Docket Center 2979_001 .pdf p. 2 Taft/ Taft Stettinius & Holiister LLP 425 Walnut Street, Suite 18001 Cincinnati, OH 45202-3457 / Tel : 533 .381 .2838 / Fax: 513 .381 .0205 Iwww.tafttaw.com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix f Beijing ROBERT A- SiLoTi 513-357-9638 bilott@taftlaw_corn June 12, 2009 FEDERAL EXPRESS EPA Docket Center, MC 2822T U .S. Environmental Protection Agency EPA West, Room 3334 1301 Constitution Avenue, NW Washington, D.C. 20004 Re: Submission to IRIS and AR-226 Database For PFOAlPFOS: EPA-HQORD-2003-0016 To IRIS Database for PFOAIPFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-933f (Feb . 23, 2006), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR226 database: 1 . Fenton, S .E ., et al., "Analysis of PFOA in Dosed CD-1 Mice Part 2: Disposition of PFOA in Tissues and Fluids From Pregnant and Lactating Mice and Their Pups," Reprod. Toxicol. (2009), doi:10.1016fj.reprotox.2009.02.012 ; 2. von Ehrenstein, O.S., et al., "Polyfiuoroalkyl Chemicals in the Serum and Milk of Breastfeeding Women," J4eprod. Toxicol. (2009), , doi:10.10161j .reprotox.2009.03.001 ; and = 3. Hines, E.P ., et al., "Phenotypic Dichotomy Following Developmental Exposure to PerFluorooctanoic Acid (PFOA) in Female CD-1 Mice : Law Doses Induce Elevated Serum Leptin and Insulin, and Overweight in MidLife," 304 Molecular & Cellular Endocrinology 97-105 (2009). 11434742 .1 CONTAINS NO CBI June 12, 2009 Page 2 RAB:mdm Enclosure . cc: Gloria Post (NJDEP)(wl encl .) (via U.S . Mail) Helen Goeden (MDH)(w! encl .) (via U.S . Mail) Lora Wemer (ATSDR)(w/ enc1 .) (via U .S_ Mail) {W1405808.1} Accepted Manuscript Title: Analysis of PFOA in Dosed CD-1 Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups Authors : Suzanne E. Fenton, Jessica L. Reiner, Shoji F Nakayama, Amy D. Delinsky, Jason P Stanko, Erin P Hines, Sally S. White, Andrew B. Lindstrom, Mark J. Strynar, Syrago-Styliani E. Petropoulou PII: DOI: Reference: S489Q-b238(09)00040-9 doi :10.10161j.reprotox .2fl09.02 .012 RTX 6230 'To appear in: Received date: Revised date: Accepted date: Reproductive Toxicology 4-2-2009 20-2-2009 25-2-2009 Please cite this article as: Fenton SE, Reiner JL, hlalcayarna SF, Delinsky AD, Stanko 3P, Hines EP, White SS, Lindstrom AB, Strynar MJ, Petropoulou S-SE, Analysis of PFOA in Dosed CD-1 Mice Part 2: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups, Reproductive Toxicology (2008), doi :10.1016Ij .reprotox.2009.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ! Analysis of PFOA in Dosed CD-I Mice Part Z: Disposition of PFOA in tissues and fluids from pregnant and lactating mice and their pups. Suzanne E. Feritor?`, Jessica L. Reine?, Shoji F. Nakayamab, Amy D. Delinsky', Jason P. Stankoa, Erin P. Hinesa, Sally S. White',", Andrew B. Lindstrom, Mark 7. Strynar`, and Syrago-Styiiani E. Petropoulou"o a Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. EPA, MD-67, Research Triangle Park, NC 2 7711, USA b fkrkridge Institutefor Science andEducation (flRISE)Research Participant, Human Exposure and Atmospheric Sciences Division, National Exposure Research Laboratory, DItD, U.S. EPA, Research Triangle Park; NC 27711, USA `Human Exposure andAtmospheric Sciences Division, National Exposure Research Laboratory, ORD, U.S. EPA, Research Triangle Park, NC 27711, USA d Curriculum in Toxicology, University ofNorth Carolina, Chapel Hill, NC 27599, USA "Current address: Division ofLaboratory Sciences, National Centerfor Environmental Health, CentersforDisease Control andPrevention, Atlanta, GA 30341, USA *Corresponding author and address: Suzanne E. Fenton, Ph.D . U.S . Environmental Protection Agency Mail Drop 67 Research Triangle Park, NC 27711 USA Tel: 919-541-5220 Fax: 929-541-4017 E-mail : fentan.snzanne("),eoa .gov 2t2012009 Page 1 of 32 .. - :~-_~.--, :YKl~,~~~:~ s~ RMia.Y~7.:Y`, i~] 2 Runuitig title : pFOA disposition in lactation Abbreviations ANOVA analysis of variance BW body weight GD gestational day LOD limit of detection LOQ iimit of quantitation ~ MS mass spectrometer PFAA perfluoroalkyl acid PFOA perfluorooetanoic acid PFOS perfluorooctane sulfonate PND postnatal day SEM standard error of the mean UPLC ultra performance liquid chromatography 212012049 Page 2 of 32 Abstraet Previous studies in mice with multiple gestational exposures to pertluorooctanoic acid (PFOA) demonstrate numerous dose dependent growth and developmental effects which appeared to worsen if offspring exposed in utero nursed from PFOA-exposed dams . To evaluate the disposition of PFOA in the pregnant and lactating dam and her offspring, time-pregnant CD-1 mice received a single 0, 0.1, 1, or 5 mg PFOA/kg BW dose (N=25/dose group) by gavage on gestation day 17. Maternal and pup fluids and tissues were collected over time. Pups exhibited significantly higher serum PFOA concentrations than their respective dams, and their body burden increased after birth until at least 8 days old, regardless of dose. Tbe distribution of milk:serum PFOA varied by dose and time, but was typically in excess of 0:20: These data suggest that milk is a substantial PFOA exposure route in mice and should be considered in risk assessment modeling designs for this compound. Key words: PFOA ; serum; amniotic fluid; urine; milk; mammary gland; dosimetry; disposition 2/20/2009 1 1. Introduction 2 Perthtorooctanoic acid (1'FOA) is a member of the perflraoroalkyl acid (PFAA) 3 family of man-made, fluorinated organic compounds used in a number of consumer 4 goods and industrial surfactants due to their grease and water-repellant properties . The. 5 use of PFAAs in many common applications, such as stain repellants for clothing,. 6 carpeting, and upholstery, and the stability ofthe carbon-fluorine bond have made them 7 ubiquitous in the environment. The predominant route of exposure in North American 8 and European consumers is likely oral intake, including drinking water, while inhalation 9 and dermal absorption comprise routes of lesser exposure [1-5]. 10 1'FAAs are persistent, readily absorbed, not known to be metabolized, and are 11 poorly eliminated, with half-lives in hurnans ranging frain roughly 4-8 years [2-4]. In 12 fact, the arithmetic and geometric mean half-lives of serum elimination, respectively, 13 were 5.4 years (95!o confidence interval-(CI), 3.9-6 .9j and 4.8 years (95!a Cl, 4.0-5 .8) 14 for PFOS ; 8.5 years (95% CI, 6.4-30.6) and 7.3 years (95/fl Cl, 5.8-9 .2) for PFHS; and 15 3_8 years (95lo Cl, 3.1-4 .4) and 3 .5 years (95% Cl, 3.0-4 .1) for PFOA [4). 16 These characteristics led to increased concern forthe potential health risks of 17 PFAAs and a program to reduce product and emission content of PFOA and related 18 chemicals was recenfly initiated [1]. PFAAs are continually detected worldwide in both 19 human and wildlife samples [3, 6-9]. A recent analysis of American Red Cross blood 20 donors indicated a reduction of 60% in blood perflus?rooctane sttlfonate (PFOS) and 25% 21 in blood PFOA levels between the years 2000 and 2006[10]. However, while the 22 production of and potential for human and wildlife exposures to certain PFAAs has been 23 reduced in the US in recent years, it is not clear that perfluorinated compounds produced 212012QQ9 1 in other countries will not continue to replace them in the US marketplace or in the 2 contribution to worldwide exposure. 3 Much of the recent health effects research on PFOA in mice, coraaamonly associated with gestational exposures of 0.01-5 ing PFOAIkg B W, has focused on 5 developmental toxicities such as decreased maternal weight gain, reduced neonatal 6 survival and body weight, as well as later life effects such as pubertal delays, mammary 7 gland abnormalities, and excessive weight gain [2, 11-161 . Earlypostnatal adverse health $ observations prompted studies examining the effect of PFOA on maternal lactation and 9 health effects of the nursing offspring. White et al. [14'] described reduced epithelial 10 differentiation on postnatal day (PND) 10 in mammary glands of CD-1 mouse dams I1 exposed to 5 mg PFOAIkg BW from GD1-17, as well as delays in epithelial involution 12 and alterations in milk protein gene expression~on PND20. In addition, female offspring 13 of exposed dams displayed stunted mammary epithelial branching and growth on PND10 14 and PND20. In a cross-foster study utilizing CD-1 mice, Wolf et al. [16] reported that 1.5 although in utero exposure to 5 nngPFOAIkg BW from GD1-17 in the absence of 16 lactational exposure was sufficient to induce postnatal body weight deficits and 17 developmental delays, pup survival from birth to weaning was affected only in those both 1$ in utero and lactationally exposed. Furthermore, recent studies [15] have shown that 19 unexposed neonates lactationally exposed to PFOA quickly developed mammary gland 20 growth deficits and that control darns nursing in utero-exposed pups (dams exposed via 21 pup groorning) demonstrated slowed differentiation of their own mammary glands that 22 was evident in whole mount preparations of the tissue by the 5d` day of lactation. These 212012009 Page 5 of 32 1 results support a role for impaired lactational development and possibly a significant 2 lactational transfer of PFOA in the observation of early growth effects. 3 The concern for potential prenatal and neonatal exposures in humans has been 4 raised further by the detection of PFAAs in human breast milk and cord blood and the 5 development-related outcomes associated with these observations . So et al. [ 17] indicated b a range of47-210 ng/L (0.047-0.21 nglznl) PFOA in 19 samples ofbreast milk from 7 Chinese women. PFOA was detected in only one of 12 human milk samples collected 8 from 1996-2004 in Sweden at a concentration of492 pglml (0.492 ng/ml; [18], and a 9 mean of 43 .8 pglml (o.Q44 rig(ml)was reported for45 U.S . breast milk samples collected 10 in 2004 [ 19]. Two studies recently determined a negative association between PFOA and 11 growth indices in children with median cord serum levels of 1.6 nglml PFOA in the U.S . 12 [20] and 5_S nglmi PFOA in Denmark [21] . While only one sample was found to contain 13 PFOA in the KarrFnan et al . [181 study, .these researchers reported a significant milk to 14 serum correlation (r2 = .7-0 .8, p<0.05) for other PFAAs detected . Furthermore, Tao et 15 al . [19] suggested that there maybe preferential partitioning of PFOA to milk compared 16 to otherPFAAs and also indicated that women who were nursing for the first time 17 exhibited 49lo higher concentrations ofPFOA inbreast milk than women who had 18 nursed previously, although inter-individual variation, daily milk output and milk protein 19 concentration were not taken into consideration . The only study that has evaluated the 24 distribution coefficient of PFCs between blood and milk in animal models was a 21 pharmacokinetic study of placental and lactational transport of PFOA in rats[22] . 22 Although female rats are known to have a serum PFOA half-life of only a few hours [23], 23 onlike mice which have a'lz-life of about 15 days [13], the study [22] indicated 212012009 Page 6 of 32 7 l concentrations in rat milk approximately 10 times less than that of maternal plasma and 2 that the milk concentrations were generally of the same magnitude as the concentrations 3 in pup plasma. 4 The increasing amount of research confirming the developmental toxicity of 5 PFAAs in animal studies, coupled with their detection in human cord blood and-imilk 6 supports the need for examining the disposition of PFAAs during pregnancyllactation in 7 an appropriate animal model in order to fully establish the association between 8 prenatal/neonatal exposure and offspring effects. While other studies have examined the 9 pharmacokinetics of PFAAs in limited contexts, little data currently exist on the 10 disposition of PFOA in pregnant and lactating mice or-their offspring. We recently 11 developed an analytical method for the analysis ofPFOA in mouse serum, urine, milk, 12 mammarytissue, amniotic fluid, and pups [24]:Utilizing these methods, we report here 13 data on the distribution of PFA in various matrices of pregnant and lactating CD-1 14 mice, as well as the senim concentration and total body load of their offspring, following 15 a single exposure of PFOA on GD 17 . These data will allow us to reduce the 16 uncertainties in risk assessment for this particular PFAA. 17 18 19 2E} 21 22 23 24 v20/2009 Page 7 of 32 1 2. Materials and methods 2 2.1 Chemicals 3 PFOA (ammonium salt ; >98% pure), used in animal exposures, was purchased 4 from Fluka Chemical (Steinheim, Switzerland) . PFOA was completely dissolved by 5 agitation in deionized tap water, in which PFOA was below the level of detection (LOD -- 6 0.5 nglL for water), and prepared fresh just prior to use. 8 2.2 .4nimals 9 A11 animal studies were conducted as approved by the National Health and 10 Environmental Effects Research Laboratory Institutional Animal Care and Use 1 L Committee. Confirmed timed pregnant female CD-1 mice (n=1U0) were purchased from 12 Charles River Laboratories (Raleigh, NC). Pregnant mice were received at the U.S. 13 EPA's Laboratory Animal Care facility on gestation day (GD) 14 (day of sperm-positive 14 designated as GDO) . Upon arrival, mice (approximately 12 weeks old) were weighed and 15 randomly distributed among PFOA treatment groups_ Tliey were housed individually in 16 polypropylene cages with Alpha-dri (Shepherd Specialty Papers, Kalamazoo, MI) 17 bedding and nesting materials. They were provided pelleted chow (I,abDict 5001, PMI 18 Nutrition International LLC, Brentwood, MO) and tap water ad libitum (both contained 19 PFOA at concentrations below the LOD). Animal facilities were controlled for 20 temperature (20-24C) and relative humidity (40-6(1%), and kept under a 14 :t0-h light21 dark cycle. Mice (r--25/dose group) received either water vehicle or a single dose (0.1, 22 1.4 or 5.0 mg/kg) of PFOA (in water, 10 pl/g) by oral gavage on GD17 . 23 2/2Q/2{?D9 Page 8 of 32 's-=='+~'k~~w'a~~ ;.-.-~~`t~N 11, ''~-s~"--,. ~ .3k: 9 1 2.3 Animal Assessments and Sample Collection 2 Live dam body weights were recorded on GD17, GD18 (prior to parturition), 3 PNDi (day after parturition), and PNDs 2, 4, 8,1 1, and 18 . On GDl8, 24 hr after the 4 PFOA exposure, five dams in each dose group were sacrificed and trunk blood, urine, 5 amniotic fluid (fluid immediately surrounding each fetus), and the 4th and 5th mammary 6 gland were collected. Liver weight, total number of fetuses (live, dead, Or resorbed), and 7 fetal weights were determined. One entire fetus from each litter was euthanized by 8 decapitation and quick frozen on dry ice in a 15 in] screwca.p vial . Remaining fetuses 9 were quickly euthanized and discarded. The dam mammary gland, urine, and arnniotic IU fluid were kept on ice, and then frozen until assayed. The~trunlc blood was allowed to 11 clot; serum was collected after centrifugation and-frozen until assayed. All samples were 12 kept frozen at -80 C. 13 A similar routine was followed on PND1 (48 hr after exposure, n=5 dams/dose 14 group) . Weights of the dam, pups, darn liver, and the number of live pups in each litter 15 were recorded. A single pup from each litter was weighed, euthanized, and quick frozen 16 in a collection vial (including all blood) . Blood from all remaining pups in each litter was 17 pooled into a single :vial, allowed to clot, and separated to serum by centrifugation. Dam SB and pup serum, dam urine and mammary tissue were frozen until assayed . All remaining 19 litters, in all dose groups, were equalized to lU pups each on PND1 . Biological samples, 20 including a single pup and pup serum, as described for PNDI, were also collected on 21 PNDs 4, .8 and 18 (n=5 dams/dose group), at the same time of day. 22 Milk collection was attempted, following administration of oxytocin (lU/ml, i .p., 23 20 min prior to milking) on both OD 18 and PND 1, but was unsuccessful. Milk was 2i2o120as Page 9 of 32 10 1 collected on I'NDs 2, 9, 11, and 18 following a 2 hr separation of the pups from the dam 2 and an oxytocin stimulus. The milk was vacuum aspirated using low, pulsatile pressure, 3 into a pre-weighed microcentrifuge tube. Collected milk was weighed and frozen until analyzed . Biological samples including urine, dam and pup serum, amniotic fluid, 5 mammary tissue, whole pup, and milk were analyzed for PFOA using the methods b described briefly below and in our companion paper [24] . 7 8 2.4 PFOA sample analyses 9 Briefly, the analysis of PFOA was performed using a Waters Aequity''N Ultra 10 Performance liquid chromatography system interfaced with a Waters Quattro Premier XE 11 triple quadrnpole mass spectrometer (UPLC-MSIMS) (Waters, Milford, MA). Either 25 12 or 50 pL of serum and amniotic fluid (50 uL used for controls), 20 wL aliquots of urine 13 and milfc, and 300 uL of pup or mammary tissue homogenates were utilized as starting 14 material for these analyses. Samples were extracted, purified, and concentrated or diluted 15 exactly as described by Reiner et at.. .[24]. 10-40 pL of the prepared sample* depending on 16 the concentration of the original exposure, was injected and run on the UPLC-MS/MS 17 [24] . Refer to Reiner et al . [24] for method performance and quality control steps that 18 were performed to insure the precision and accuracy of the methods used. The limit of 19 quantitation~(LOQ) for these experiments were 5 ng/ml (serum), 1 nglml (amniotic fluid, 20 urine, milk); and 1 ng/g (whole pups, mammary tissue). 21 22 23 212t112009 Page 10 of 32 a~x. ^.r-J ~'''--'~ 11 1 2.5 Urinary ereatinine measures 2 Creatinine concentrations were measured as a basis to evaluate PFOA in mouse 3 urine. The QuantiChrom creatinine assay (BioAssay Systems, Hayward, CA) exhibited 4 an LOD of 0.10 ngiml andwas linear up to 300 ng/ml. Thirty pl of each urine sample was 5 prepared and evaluated at 510 nm singly or in duplicates (five duplicates per set of 20 6 samples) according to the manufacturer's instructions. The inter-assay coefficient of 7 variation (CV) ranged from 4.0-6 .8lo and the intra-assay CV ranged from 0.3-16.1 lo, 8 with an average of 4.9l0. The assay standard accuracy ranged from 0.2-8 .4lQ. Urinary 9 PFOA is reported as corrected for creatinine concentrations '(ng PFOA1g creatinine). 10 11 2.6 Computations and Statistics 12 Reported PFOA concentrations have been adjusted for dilution or concentration 13 factors, as well as creatinine levels (ng"g; urine), or the weight of the tissue evaluated 14 (nglg; mammary tissue and whole pups). Serum, amniotic fluid, milk and Urinary 15 concentrations are reported as .nglml . Averages, proportions, and statistical comparisons 16 were calculated with SAS 9.1 (SAS Institute; Cary, NC). Statistical significance was 17 determined using a Proc GLM ANOVA, with a 1}unnett's post-hoc comparison, and 18 significance was set at p<0.05. 19 20 21 22 2120l2009 Page 11 of 32 12 1 3. Results 2 3.1 Biological Outcomes 3 This is the first study to report single dose disposition of PFOA in pregnant and 4 lactating mice and their offspring. The doses chosen were based on previous reports in 5 CD-1 mice [3,14, lb] demonstrating developmental health outcomes following multiple 6 gestational PFOA exposures. A single PFOA exposure on GD17 did not affect the 7 number of live fetuses (on GD18), implantation sites, or live-born pups (on PNDI), or 8 dam body weights (data not shown). Unlike studies using multiple gestational PFOA 9 exposures [13, 25], there was no change in pup body weight, dam liver weight, and dam 10 liver:BWratios, within the PFOA dose range administered in this study (Figure 1). The 11 rise in dam liver:BW ratio between GDI8 and PNDI., which persisted until weaning, was 12 due to the dramatic decrease in body weight at parturition, as this single late gestation 13 PFOA exposure failed to change mean liver weight in exposed dams, compared to control 14 values, at any time evaluated. 15 16 3.2 PFOA Concentrations Prior to Birth 17 The mean concentration of PFOA in the amniotic fluid and serum of the exposed 18 dams 24 hr after exposure is shown (Figure 2; amniotic fluid controls average 3.8 ng(ml) . 19 The average concentration of PFOA detected in dam serum was about twice the amniotic 20 fluid concentration at eacb dose evaluated (amniotic fluid was 68 .8, 51 .8, and 40ta of 21 dam serum levels at 0.1, 1, and 5 mg PFOAIIcg BW, respectively). A comparison of the 22 amount of PFOA in an entire GD1 B fetus (body burdenlpupstandard error of the mean . 23 [SEM]; Figure 5) to the GD18 PFOA concentration in amniotic fluid (nglrnl ; assuming 1 2120/2009 Page 12 of 32 13 1 ml total volume) reveals 2.3-, 3.l-, and 2.7-fold increased PFOA in the pup vs. the fluid 2 in which it was contained in utero for 0.1, 1, and 5 mg/kg dose groups, respectively. 3 4 3.3 PFOA Concentrations in the Dams 5 The serum and urine PFOA concentrations were evaluated in dams that were 6 nursing litters of approximately 10 pups (pNDI equalized; minimal pup loss over time). 7 As expected, dam sera contained the highest PFOA concentrations of any matrix 8 evaluated, regardless of dose (Figure 3 ; all serum controls <LOQ). The rise in circulating 9 serum PFOA with increasing dam exposures was proportional to the change in dose 1(l delivered, regardless of stage of lactation (i.e., mean 9.9 fold and 5.I-fold increases l l between 0.1-1 .0 mg/kg and 1 .(}-5.U mg/kg exposures, respectively). 12 A one-time PFOA exposure of 0.1 mglkg produced an average darn serum PFOA 13 concentration (Figure 3A) of 144-226 ng/ml at 24 and 48 hr after exposure, respectively, 14 which was reduced to an average of 44 nglml near the peak of lactation (pND8), and had 15 risen to a mean of 123 nglml by PNDJ 8, a time when the pups' primary caloric intake 16 came from rodent chow and not milk. The U-shaped serum concentration curve observed 17 in the 0.1 mg PFOAakg dose group was also present in the l and 5 mg/kg exposure 18 groups. 19 As shown in Figure 3 (A-C ; control urine and mammary gland PFOA <LOQ), 20 although the concentrations of PFOA cannot be compared directly between serum, urine, 21 and mammary tissue, due to the difference in units, it was evident that much less PFOA 22 was being excreted in dam urine than was present in dam serum, and that mammary 23 tissue contained a considerable amount of PFOA . While a U-shaped response in dam 2no/2o09 Page 13 of 32 -.--=.xm.i-,n. r -;'.wsr~:4~'a`~s 14 I excretion of PFOA (urine) was not as pronounced as that of serum, mammary tissuc 2 demonstrated a strong U-shaped response, with the lowest concentrations measured near 3 the peak of lactation, and a significant rise in concentration apparent again at PND 19 4 (p<0.05)- 5 When aspirated milk PFOA values were evaluated (Figure 3D; 1 control >LOQ), - b a U-shaped curve over time was again evident. As depicted in Table l, the percentage of 7 PFOA in milk (compared to serum) was substantial . Comparing the milk concentrations 8 to the closest matched dam serum concentrations {by time}, the amount -of PFOA in the 9 milk consistently ranged from If10 to 112 that of dam serumt~PFOA across dose and time . 10 It appeared that the day of lactation on which milk PFOA-was measured had an important I 1 influence on this relationship. The milk:serum PFOA ratio was greatest in early and late 12 lactation (PND2 and PND18), ranging from 15-5b% (means of 33% early and 2b/a late), 13 while near the peak of lactation (PND8 and 11), the PFOA milk :serum ratio ranged from 14 11-27% (mean 1'1-7%). It was not possible to accurately measure the volume of milk 15 obtained at aspiration, but precise weights were compared . On PNDs 2, 8, 11, and 18, the 16 average weight of milk obtained via aspiration of control mice was 0.072, 0_1906, 17 0.2547, and 0.0457 g, respectively, demonstrating over a 3.5-fold increase in weight from 18 PND2 to I I and a 5.6-fold drop from PND11 to 18 . 19 - 20 3.4 PFOrI Concentrations in the Pups 21 Pup serum PFOA concentration was evaluated on PNDs 1, 4, 8, and 18 . In 22 comparing the average PFOA concentrations in PNDI pups vs . their respective dams 23 (Figure 4A; whole control pups and control serum < LOQ), it appeared that circulating 2/20J2009 Page 14 of 32 .;: ~.. - t.~. . _-- --tt'~A.w p. 19 15 1 pup serum PFOA concentrations were significantly higher than those measured in dams, 2 regardless of dose (p<0 .05). Although pups possessed a substantially higher serum PFOA 3 concentration than dams, the difference in pup and dam blood volumes at those stages of 4 pup development are considerable. Regardless of those differences, heightened 5 circulating PFOA in pup sera reflected increased exposures, proportional to dose 6 throughout lactation (i .e., mean 10.4-fold and 4.3-fold increases between 0:1-1 .0 mg/kg 7 and 1 .0-5 .0 mg/kg exposures, respectively). 8 Unlike their dams, pups did not demonstrate U-shaped serum PFOA 9 concentration curves (Figure 4B). Pup serum PFOA concentrations continued to exceed IO the average dam serum PFOA concentrations over time, until PND18 when the pup and 11 darn concentrations approached 1 :1 . When the PFt7A concentration (ng/g) was evaluated 12 in whole pups (pup and blood; Figure 5 left panels), a decline in PFOA concentration was 13 detected over time, across all doses. However, when the rapidly increasing body weight 14 of the pups was taken into consideration to calculate the total amount of PFOA in the 15 neonate (as shown in Figure 1), a cornpletely different trend was noted (Figure S right 16 panels). Regardless of exposure dose, PF{}A body burden (adjusted for weight) rose I'I through the peak of lactation and had begun to decline by PNDI 8, demonstrating an 18 inverse U-shaped curve. When the administered PFOA dose and measured body burden 13 in whole pups ; (body weight taken into effect) were compared the administered 20 PFOA :measured PFt3A ratio was no longer proportional throughout lactation, and unlike 21 the ratios reported for dam and pup serum PFOA. Mean body burden ratios of 13 .2-fold 22 (range 11 .1-1'7 .8) and 4.3-fold (range 3.2-5 .1) increases between 0.1-I .) mg/kg and 1 .023 5.0 mg/kg exposures, respectively, were determined . 24 2120l2009 Page 15 of 32 ...~,.L.v- 16 1 4. Discussion 2 These data confirm that on a concentration-based comparison, gestationally 3 PFOA-exposed pups exhibited a significantly larger serum PFOA load than their dam . 4 That substantial serum PFOA load in pups was evident 24 hr after a single exposure, and S was apparently due to blood-borne (transplacental) transfer. Another important discovery 6 is the U-shaped PFOA concentration over time, regardless of dose, in the dam mammary 7 tissue, milk, and serum. This unique PFOA response was not detected in pups or pup 8 serum, and was evident to a lesser extent in the dams' urinary excretion curves. However, 9 when PFOA body burden in whole pups was the unit of measure, an inverse U-shaped 10 curve was apparent, and the PFOA burden of pups is proposed to increase due to milk- 11 bome PFOA intake. 12 The decline in concentration seen in the milk, mammary and serumU-shaped 13 curves is hypothesized to be due to hydro-dilution associated with increased blood and 14 milk volumes. Several physiological conditions are changed during lactation that have 15 been well documented in rats and directly relate to mice as their lactation period is of the 16 same length . A decrease in total plasma proteins due to increased blood volume, cardiac 17 output, and blood flow to certain tissues, such as the mammary gland has been reported 18 in rats [26, 27]. Elevated blood volume is due to increased plasma volume [27] . Milk 19 yield {g/hr) in-rats was reported to reach its peak by PND 10 [271 and the rat mammary 20 gland reaches its maximum size (as % body weight) by PND15 [26], with a steep rise in 21 size from PNDS-1S. Rat mammary gland blood flow and volume of milk produced are 22 directly related, when measured on PND15 (2b] . Total serum proteins are lower in 23 lactating rats that those measured in non-lactating rats [27], and in humans, serum 212fl12fl09 Page 16 of 32 17 1 albumin concentration decreases during pregnancy and early lactation [28] . Further, at 14 2 d postpartum, the cardiac output of lactating rabbits was 3to higher than that in non- 3 lactating animals, and the mammary gland was the only organ shown to increase in 4 weight, relative to body weight [29] . 5 Although a complete set of data that could address the exact reason for the U- 6 shaped curves during lactation was not collected in this study, the aspirated milk weights 7 did reveal a dramatic increase in milk volume (assumed due to weight change) from S PND2 up to the peak of lactation (PND11), This dramatic change, in volume (weight) 9 may explain the decrease in milk PFOA concentration seen-between PND2 and PNDI 1 . 10 PFOA also appears to concentrate in serum and milk near.xheend of lactation (PND18, I l for example) when pups are eating more chow and suckle less often. Mammary gland 12 blood flow has been reported to decrease by half in a 241u period, when suckling rat 13 offspring are removed from the dam [26j, and this fail in mammary blood flow is directly 14 associated with decreased cardiac output and % blood flow used by the mammary gland. 15 In this study a precipitous drop in weight ofmilk collected between the peak oflactation 16 and PND18 was noted, indicating-a rapid decrease in milk volume. Therefore, the U- 17 shape of the dam PFOA curves are proposed to be driven by physiological dilution and 18 concentration of the PFOA load over the period of lactation, reaching the greatest dilution 19 at or near-the peak of lactation when the milk volume produced by the dam and 20 consumed by- the pups is the greatest . Increased consumption of milk up to PNDI1 likely 21 directly contributed to the accumulation of body burden in the pup over this life stage. 22 A significant contribution of milk-borne 1'FOA transfer in CD- I mice was 23 detected in these studies. Previous reports in rats j22} and humans [ 18] have estimated 212(1f2Q09 Page 17 of 32 . . .__--. '~-' 18 1 that the dam PFOA milk:serum distribution ratio was .1 and 0_01, respectively . In the 2 present study, the distribution ratio ranged from slightly more than 0 .5 to t? . 1 in mice, 3 depending on dose, with the lowest doses tested demonstrating the highest ratios over 4 time . If the milk PFOA concentrations had been measured near the peak of lactation only 5 (days $-11), the 0.1 milk :sera distribution estimate previously reported for rats in mid6 lactation (22j may have also been presumed true in mice . However, at two periods during 7 lactation (early and late) spikes of increased milk:serum ratios appeared, regardless of 8 dose, with a substantial peak in milk PFf7A concentrations on pND2 . Although volumes 9 of milk large enough to perform analytical measures prior to PND2 were not able to be 10 obtained, we suspect, based on the significant PFOA concentrations in the PND1 ll mammary gland, that substantial milk PFOA concentrations would have been evident on 12 PNDI, as well, primarily due to being condensed in small milk volumes. 13 In previous reports by Lau (13J, Wolf [16], White [14j and co-workers, decreased 14 body weight gain and neonatal mortality were evident on several days just after birth in 15 CD-1 mouse litters gestationatly exposed to 3 mgfkg PFOA and higher, In fact, in a 16 . cross-foster study (16) demonstrating decreased body weight gain at 5 mglkg from in 17 utero exposure only, significant decreases in body weight gain were detected in the 3 1$ mg/kg dose group only when in utero exposed mice were also allowed to nurse from a 19 PFOA exposed dam. Even at 5 mg/kg, there was no evidence of decreased pup body 20 weight or neonatal mortality in the current study, following a single gestational PFOA 21 exposure . Our PFOA measurements in whole pups indicate that the PFOA body burden 22 accumulates in early life, and begins a decline as pups mature, open their eyes, and begin 23 to eat chow and drink water. Our data and those demonstrating deleterious health 212012009 Page 18 of 32 19 1 outcomes suggest that the milk of gestationally PFOA-exposed mice was a major source 2 of continued exposure to this compound for the developing pups. 3 As expected, large differences in dam and pup serum PFOA concentrations from 4 those previously reported [ 14, 16] were noticed, and those differences bring to light the 5 issue of single vs . multiple dose kinetics. As noted for PFOS, single dose kinetics may differ substantially from those involving repeated doses [30] . Concentration dependent 7 changes in clearance can result in discrepancies between single and repeated dose 8 kinetics . 9 A limited number of epidemiological studies have revealed associations between 10 health outcomes (birth weight, head circumference) and cord-blood or maternal serum PFOA concentrations in humans [2{l, 21j, while other studies failed to detect associations 12 with later developmental milestones in infants ~[31]. Several studies have now measured 13 PFAAs in human milk [17-19, 32, 33], however only one study has been able to 14 approximate the milk:serum relationship of PFOA transfer [18] . The reported 0.01 15 {1110(ye) relationship was determined from a single voluntarily contributed sample at 3 16 weeks postpartum. According to the mouse miik:serum PFOA distribution over time that 17 we report herein, the values reported in one human [18] and rats [22] may not be 18 representative of the PFOA distribution to milk throughout lactation in those species. 19 In conclusion, these studies confirmed and further defined considerable PFOA 20 exposures to mouse offspring following a single gestational exposure. They also 21 demonstrated the accumulation of chemical over time in whole pups, which likely results 22 from milk-borne PFOA, an exposure that had previously been incompletely assessed in 23 other species. A single 0.1 mg/kg PFOA exposure to apregnant mouse induced 212012009 Page 19 of 32 zg 1 circulating serum PFOA concentrations of 44-216 nglml in darns and 117-326 nglml in 2 pups ; values similar to or lower than serum PFOA concentrations of children that were 3 accidentally exposed via DuPont production plant emission (34], Because of evidence 4 [15, 35] demonstrating neonatal and latent health effects following developmental 5 exposures to PFOA in mice, associated with higher circulating PFOA levels than those 6 reported here, continued studies evaluating exposure-effect relationships are warranted in 7 children. $ 9 10 11 12 13 Acknowledgexnents 14 The authors would like to thank Drs. Bar-bara Abbott (US EPA, Reproductive Toxicology 15 Division) and Chester Rodriguez (National Center for Computational Toxicology, US 16 EPA) for their constructive criticisms of this manuscript. We acknowledge the excellent 17 care of these animals by New Year Tech, Inc. (Restin, VA). The research in this article 18 has been reviewed by the National Health and Environmental Effects Research 19 Laboratory, US Environmental Protection Agency (EPA), and approved for publication. 20 Findings in this report are those of the authors and approval does not signify this report 21 reflects EPA policy . The use of trade names or commercial products does not constitute 22 endorsement or recommendation foruse. 23 24 2/2012009 Page 20 o4 32 21 1 References 2 [1] U.S . Environmental Protection Agency, Announcement of the 20101151'FGIA 3 Stewardship Program by Administrator Stephen L. Johnson. (2006) Available at 4 http:llwww.epa.govlopptintrlpfoalpubslpfoastewardship .htm. Accessed 21312009. 5 6 [2] M.E. Andersen, J.I.. Butenhoff, S.C. Chang, D.G. Farrar, G.L. Kennedy, Jr., C. 7 Lau, G.W . Oisen, J. Seed and K.B. Wallace, Perfiuoroaikyl adds and related chemistries8 -toxicokinetics and modes of action, ToxicolSci,102 (2008), 3-14 . 9 10 [3] C. Lau, K. Anitole, C, Hodes, D. Lai, A. Pfahles-Hutchens and l. Seed, 11 Perfluoroalkyl acids: a review of monitoring and toxicological findings, Toxicol Sci, 99 12 (2007),366-394~ 13 14 [4] G.W. Olsen, J.M. Burris, D.J . Ehresman, J.W. Froehlich, A.M. 5eacat, J.L. 15 Butenhoffand L.R. Zobel, Half-life of serum elimination of perfluorooctanesulfonate, 16 perfluorohexanesulfonate, and perfluorooctanoate in retired #7uorochemical production 17 workers, Environ Health Perspect,115 (2007), 1298-E3fl5. 18 19 [5] D. Trudel, l.. Horowitz, M . Worrnuth, M.:Scheringer,l .T. Cousins and K, 20 Hungerbuhler, Estimating consumer exposure to PFOS and PFOA, Risk Anal, 28 (2008), 21 251-269. 22 23 [6] K.S. Guruge, P.M. Manage, N. Yamanaka, S, Miyazaki, S. Taniyasu and N. 24 Yamashita, Species-specific concentrations of perfluoroalkyl contaminants in farm and 25 pet animals in Japan, Chemosphere, 73 (.2008), S210-215 . 26 27 [7] G.W. Olsen, H.Y . Huartg, K.J. Helzlsouer, K.J. Hansen, J.L . Butenhoffand J.H . 28 Mandel, Historical comparison of perfluorooctanesulfonate, perfluorooctanoate, and 29 other fluorocheEnicals in human blood, Environ Health Perspect,113 (2005), 539-545. 30 31 [8] L. Tao, K_ Kannan, N. Kajiwara, M.M . Costa, G. Filimann, S. Takahashi and S. 32 Tanabe, Perfluorooctanesulfonate and related fluorochemicals in albatrosses, elephant 33 seals, penguins, and-polar skuas from the Southern Ocean, Envirorz Sci Technol, 40 34 (2006),7642-7648 . 35 36 [9] L.W, Young, M.K . So, G. Jiang, S. Taniyasu, N. Yamashita, M: Song, Y. Wu, J. 37 Li, J.P, Giesy, K.S . Gurage and P.K. Lam, Perfluorooctanesulfonate and related 38 fluorochemieals in human blood samples from China, Environ Sci Technol, 40 (2006), 39 715-720. 40 41 [101 G.W . Olsen, D.C . Mair, T.R. Church, M.E. Ellefson, W.K. Reagen, T.M . Boyd, 42 R.M. Herron, Z. Medhdizadehkashi, J.B . Nobiletti, J.A. Rios, J.L . Butenhoffand L.R . 43 Zobel, Decline in perfluorooctanesulfonate and otherpolyfluoroalkyl chemicals in 44 American Red Cross adult blood donors, 2000-2006, Environ Sci Technol, 42 (20D8), 45 4989-4995. 2r20f2009 Page 21 of 32 22 1 2 111) J.L. Butenhoff, D.W. Gaylor, J.A. Moore, G.W. f3lsen, J. Rodr'scks, J.H. Mandel 3 and L.R. Zobel, Characterization of risk for general population exposure to 4 perfluorooctanoate, Regul Toxicot Pharmacol, 39 (20U4), 363-380. 5 6 [12] C. Lau, J.L. Butenhoffand J.M . Rogers, The developmental toxicity of 7 perfluoroalkyl acids and their derivatives, ToxFcolAppl Pharrnacol,198 (2004), 231--241 . 8 9 [13) C. i.au, J.R. Thibodeaux, R.G. Hanson, M.G. Narotsky, J.M. Rogers, A.B. 10 Lindstrom and M.J . Strynar, Effects of perf)uorooctanoic acid exposure during pregnancy 11 in the mouse, ToxicolSci, 90 (2U06), 510-518. 12 13 [14] S.S. White, A.M. Calafat, Z. Kuklenyik, L. Villanueva, R.D. Zehr, L. Helfant, 14 M.J . Strynar, A.B . Lindstrom, J.R . Thibodeaux, C. Wood and S.E. Fentoa, Gestational 15 PFOA exposure of mice is associated with altered mammary glaiid development in dams 16 and female offspring, Toxicol Sci, 96 (2007),133-144 . 17 18 [15] S.S_ White, K. Kato, L.T . lia, B.J . Basden, A.M. Calafat,~E.P, Hines, J.P . Stanko, 19 C.J . Wolf, B.D. Abbott and S.E. Fenton, Effects of perfluorooctanoic acid on mouse 20 mammary gland development and differentiation.resulting from cross-foster and 21 restricted gestational exposures, Reprod Toxicol (2p08) . 22 23 [16] C.J. Wolf, S.E. Fenton, J.E. Schmid, A.M. Calafat, Z. Kuklenyik, K.A. Bryant, J. 24 Thibodeaux, K.P. Das, S.S . White, C.S . Lau and B.D . Abbott, Developmental toxicity of 25 perfluorooctanoic acid in theCD-1 mouse after cross-foster and restricted gestational 26 exposures, ToxacolSci, 95 (20Q7), 462-473. 27 28 [17] M.K . So, N. Yarnashita,, S. Taniyasu, Q. Jiang, J.P . G'sesy, K. Chen and P.K. Lam, 29 Health risks in infants associated with exposure to pertluori,nated compounds in human 30 breast milk from Zhoushan,:China, Environ Scf Technol, 44 (2006),2924-2929 . 31 32 (181 A. Karrman, I. Ericson, B: van Bavel, P.O. Darnerud, M. Aune, A. Glynn, S. 33 Ligaell and G. l.indstrorn, Exposure of perfluorinated chemicals through lactation: levels 34 of matched human milk and serum and a temporal trend, 1996-2fl4}4, in Sweden, Environ 35 Health Perspect,115 (2007), 226-230. 36 37 [19] L. Tao, K. Kannan, C.M . Wong, K.F. Arcaro and J.L . Butenhoff, Perfluorinated 38 compounds in human milk from Massachusetts, U.S .A, Environ Sci Technol, 42 (2048), 39 3096-3101 . 40 41 [20] B.J . Apelherg, F.R . Witter, J.B . Herbstman, A.M. Calafat, R.U . Halden, L.L. 42 Needham and L.R . Goldman, Cord serum concentrations of perfluorooctane sulfonate 43 (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth, Environ 44 Health Perspeci,115 (2{l(17), 167fl-1676. 45 2J2012009 Page 22 of 32 23 1 [21] C. Fei, J.K McLaughlin, R.E. Tarone and J. Olsen, Fetal growth indicators and 2 perfluorinated chemicals: a study in the Danish National Birth Cohort, Am JEpidemiof, 3 168 (2008), 66-72. 4 5 [22j P.M. Hinderliter, E. Mylchreest, S.A. Cannon, J.L . Butenhoff and G.L. Kennedy, 6 Jr., Perfluorooctanoate: Placental and lactational transport pharmacokinetics in rats, 7 Toxicology, 211(2005),139-148 . 8 9 [23] J.P . Vanden Heuvel, B.I . Kuslikis, M.J. Van Rafetghem and RE. Peterson, Tissue 10 distribution, metabolism, and elimination of perfluorooctanoic acid in male and female 11 rats, JBiochem Toxicol, 6 (1991), 83-92. 12 13 [24] J.L . Reiner, S.F. Nakayama, A.D . Delinsky, J.P. Stanko, S.E. Fenton, A.B . 14 Lindstrorn and M.J. Strynar, Analysis of PFOA in dosed CD 1 mice: Part. 1. Methods 15 development for the analysis of tissues and fluids from pregnant and lactating mice and 16 their pups, Reprod Toxicol (2008). 17 18 [25j B.D. Abbott, C.J. Wolf, J.E. Schmid, K.P. Das, R.D. Zehr, l,. i-lelfant, S. 19 Nakayama, A.B_ Lindstrom, M.I . Strynar and C. Lau, Perfluorooctanoic acid induced 20 developmental toxicity in the mouse is dependent on expression of peroxisome 21 proliferator activated receptor-alpha, Toxicol Sci, 98 (2007), 571-581 . 22 23 [2fi] A. Hanvvell and J.L . Linzell, The effects of engorgement with milk and of 24 suckling on mammary blood flow in the rat, .IPhysiol, 233 (1973), 111-125. 25 26 [27] K. Suzuki, H. Hirose, R. Hokao, N. Takednura and S- Motoyoshi, Changes of 27 plasma osmotic pressure during lactation in rats,J Vet Med Sci, 55 (1993), 561-564. 28 29 [28] M. Dean, B. Stock, R.J . Patterson and G. Levy, Serum protein binding of drugs 30 during and after pregnancy in humans, Clin Pharmacol Ther, 28 (1980), 253-261 . 31 32 [29J C.S . Jones and D.S. Parker, Mammary blood flow and cardiac output during 33 initiated involution of the mammary gland in the rabbit, Comp Biochem PhyslolA Comp 34 Physfnl, 91 (1988), 21-25. 35 36 [30] T..A. Harris and H.A. Barton, Comparing single and repeated dosimetry data for 37 perfluorooctane siilfonate in rats, Toxicol Lett,181 (2008),148-156 . 38 39 [31] C. prei, J.K. IvlcLaughlin, R.E. Tarone and). Olsen, Perfluorinated chemicals and 40 fetal growth: a study within the Danish National Birth Cohort, Envlron Health Perspect, 41 115 (2007),1677-1682 . 42 43 [32] W. Volkel, O. t'renzel-Boroviczeny, H . Demmelmair, C. Gebatter, B. Koletzko, 44 D. Twardella, U. Raab and F-1. Fromme, Perfluorooctane sulphonate (PFOS) and 45 perfluorooctanoic acid (PFOA) in human breast milk : results of a pilot study, Int JHyg 46 Environ Health, 211(2fl08), 44d-446. 2r2012009 Page 23 of 32 24 1 2 [33) 4.S . von Ehrenstein, Fenton, Suzanne E., Kato, Kayoko, Kuklenyik, Zsuzsanna, 3 Calafat, Antonia M., Hines, Erin p. , Polyfluoroalkyl Chemicals in the Serum and Milk of 4 Breas#feeding Women Reprod Toxicot, In Press (2009). 5 6 [34j E.A. Emmett, F.S. Shofer, H. Zhang;D. Freeman, C. Desai and L.M. Shaw, 7 Community exposure to perfluorooctanoate : relationships between serum concentrations 8 and exposure sources, J Occup Environ tY1ed, 48 (2006), 759-774 . 4 10 [35] E.P . Hiues, White, Sally S., Stanko, Jason P., Gibbs-Floumoy, Eugene A., i.au, 11 Christopher, Fenton, Suzanne E., Phenotypic Dichotomy Following Developmental 12 Exposure to Perfluorooctanoic Acid {PFDA} in Female CD-1 Mice: Low Doses Induce 13 Elevated Senun Leptin and Insulin, and Overweight in Mid-life, Molec Cell Endocrinol, 14 In Press (2009) . 15 16 17 212012009 Page 24 of 32 25 1 Fieure letrends: 2 Figure 1. Dam tissue weights and average pup weights following a single gavage PFOA 3 exposure on GD17. PFOA was without effect on several biological end points (p>0.05), 4 such as dam body weight measured on several postnatal days (PND) and on gestation day 5 (OD)IS (not shown). (A) Dam liver weight, (B) liver:body weight ratio, and (C) .pup 6 body weight over time or numbers of live pups or fetuses (not shown) were'aIso 7 unchanged by a single 1'FOA exposure . Data are shown as Mean SEM or as a mean S ratio. 9 Figure Z. Comparison of gestation day (GD)18 dam serum and amniotic fluid PFOA 1 0 concentrations. PFOA concentrations were significantly higher in dam serum than I 1 an3niotic fluid at all doses evaluated (p<0 .tY5). Data are shown as Mean + SEM. 12 Figure 3. PFOA concentrations in exposed dams . PFOA concentrations were measured 13 in dam serum (A ; ng/ml), urine (B ; nglg e=eatinine), and mammary tissue (C ; ng/g tissue 14 weight) on postnatal days (1'ND) 1, 4, 8 and 18. PFOA concentrations were measured in 15 aspirated milk samples collected on .FNDs 2, 8, 11, and 18 (D; ng/mi) . Although panels 16 A-C and B-D cannot be directly compared (due to different units), the U-shaped 17 concentration curve present in dam serum (regardless of dose) was also detected in 18 mammary tissue and aspirated milk. Data are shown as Mean + SEM. tDenotes a single 19 reliable measurement at this time due to insufficient volumes in other dams at this dose 20 and time. 21 Figure 4. Neonatal transfer of PFOA to pups. (A) A significantly higher PFOA 22 concentration in pup vs . dam serum on PNDl was noted (p<0.45; v:v). (B) Pooled pup 23 serum PFOA concentrations did not demonstrate a U-shaped curve, but gradually 2/2fll2004 Page 25 of 32 26 1 declined over time, presumably due to dilution of dose by increased growth-related blood 2 volume . Data are shown as Mean f SEM. 3 Figure 5. Whole pup PFOA concentrations. PFOA concentrations were measured in a 4 representative whole pup (pup and blood; nglg; left panels) from each litter. Although 5 there is a consistent downward trend in PFOA concentration over time, the rapidly 6 increasing blood volume and body weight changes must be taken into consideration when ? interpreting these data . Body weight-adjusted values (right panels; [nglg FFOA $ measures*g body weight = body burden]) demonstrate an accurnulation of exposure until 9 late in the lactational period. Data are shown as Mean SEM. IQ 11 12 2v2o/2o09 Page 26 of 32 Figure 1 . 3.6 3.0 2.5 2.0 1 .5 1 .0 0.5 0.0 8 0 r s m (A) Dam Liver Weight Control 0.1 1 PFOA Exposure (mgikg) (B) Dam Liver:BW Ratio 4 Control 0.1 1 5 PFOA Exposure (mglkg) (C) Pup Body Weight 10 8 6 4 2 0 Control 0.1 1 5 PFOA Exposure (mgllcg) GI GQ18 E3 PND1 p PND4 i"d PND8 UPHD18 Page 27 of 32 Figure 2 . 9000 8000 E 7000 6000 a 5000 0 4000 3000 2000 1000 0 0 .1 1 5 PFOA Exposure (m9Ng) Page 28 vi 32 p. 33 OEM Figure 3. 200 150 ti C O 10D fL Q SO 0.1 m9ft \ GD10 YND1 MIN PNDB PND1B C 20 C R +5 ~ af W 10 D.1 mglkg O 5 0 . a. PNDS PND2 PND4 PM18 PND18 2500 2000 1500- 1000 100 140 120 100 . 80 00 40 -1 20 " D- (A) Dam Serum PFOA i mglkg 12ooa 50000 8000 ~ 6000 1 4000 ~' 2000 ~ t GD16 PND1 PND4 PNDB PND1& (B) Dam Urine PFOA 0 1 -9&9 400 200 . PNDt PNM PND4 PNDB PNDts !11 I'!h 5 mglkg 5^~ `L\ n~ MSS PHD1 PM4 PNDS PN016 5 mglkg " ( ~. PNDS PND2 PN04 PND& PND18 S 2 a +3 ~- tu 0.1 mgllcg (0) Dam Mammary Gland PFOA 200 - y mBlkB 300 1590 21W loao \. 100 GD+S PNDt P?i04 PNDB PND+S GD18 PNO1 PND4 PNDt PND1L 5 ~ngikg t\: .1 1 1 }~~ V4-('i2 GD18 P=I PN04 PNDa PND+B 40 L 50 a 0 20 D. 1000 Q.1 Rtatkg goo (D) Dam Milk PFOA 3004 - 7 Ri9~9 2500 - t 604 ~ 1300 400- ion 200 ~ soo 5 0tc111c9 PN02 PND9 PND11 PND18 PND7 PNDB PNO++ PNDiS Time PND2 PNDB PND+1 PND18 Page 29 of 32 Figure 4. (A) Dam vs. Pup Serum PFOA 404 al 300 . r. Q 44Q :, . rf- 2 104 I H o CIA i PFQA Exposure (mglk9) (B) Pup Serum PFOA Over Time D.1 m9 k9 I ff 9 k8 ,8404 , 1000 . 200Q' Postnatal Day (PND) S mglkg 38 Page 30 of 32 ME " ' ~7~-,~ "vnR:~T"V)~T~LsJt.~,vy,s : ~ '1. '~i'~1 Figure 5. A- 10000 800Dr soool at~00 2000 3, r-xn . t,i~ 5 mg/kg o 6019 1 4 8 18 2000 GDi8 1 4 8 18 20000 '1s000 -.c.0.) a I` 12000 a ~ 8000 m .4000 .~ m 0 0 5000 _ 1500 d0 100D LL IL [ 4000 v 4 O ' 3000 I1 C d 2000 = EO a V '1000 C. 180 140 at c ' 100 0.1 mglkg ~0 400 a 60 20 \\" 0 POI GD78 1 4 8 18 Gestation (GD) and Postnatal (PND) Day of PFOA Measurements Page 31 of 32 O E E ~ N m 0 0~ o L ~Qa F c^J N r 0 0 a. W c. fl E oE o 0 O ms ~a aQ r r Cm r z 4 u z A. 3 O m Q DD CD li. b O n. _ VI Yr CE c U L ~< G iT 6a1 . Q . N u. 4 0 e' a V E o C r CL. Q. ! O O9" E ~ t G. G . Q m m aL'." ~ =D F^ Va C? m c S~j 0 C7 \ r 0o E 0 +~ C . w CK 0 p M t~ E 0 e- R u T \ o N m ou~Q. 160. m L a 'a m E o ~m ~E e m ~a Y0 m 'co C O ~' O =A. +' 4 "C1 0-0 Ct'C 11 ZO R O 4 Og~, ~w m w 0 Im 0 > n y C SS. tp O 7 L VI~ n p R tp O L ~ u% o. cs s ~r aCO smo p. 37 Reproductive Toxicology xxx (20D95 xxx-xxx "~ CC' ` i" journal homepage'.Www .elsevier~comilocate/r.ac.protox. , tz1 P.olyfluoroalkyl chemicals in the serum and milk of breastfeeding women z Ondine 5 . von Ehrenstein a~`, Suzanne E . Fenton b, itayoko Itatoc, 7stlzsanna Kuklenyikc, Antonia M. Calafatc, Erin P: Hinesb"' 9 'UCLA ScMool OfPublic Health. University ofCaljfamia, Los Angeles, CA, United Scares 5 5 US Enviranmenral Protection Agency, ORd, NHEERt, Reproductive ToXttotogyt3ivLsfan, Krf; NC, llafred Stares s t Centrrs fur Disease Connvf S' Prevention. Division ajl.aboratory Science, National Centerjor Environmental Health Arfortra . GA, United Stores a A RT I C LE I N F O a to Artfde history. r, Received 28 January 2009 $z Received in revised form 27 Febnea ry 2009 ra Accepted 2 March 2009 14 Available online xxx is +s Keywords: 1I 7 Palyfluoraatkytdhemicals e Pertluoroalkyl acids +s Pertluorooctanoic acid 20 Pertluoroocrane sulfonic acid zr Serum rs Breast milk 2a Lactation ABSTRACT Polyfluoroafkyl chemicals (PFCs) comprise a group of man-made organic compounds. some of which are persistent contaminants with developmental toxicity shown in laboratory animals. There is a paucity of human perinatal exposure data. The US EPA conducted a pilot study (Methods Advancement in Milk Analysis) including 34 breastfeeding women in North Carolina. Milk and serum samples were collected at 2-7weeksand 3-4 months postpartum : 9 PFCs were assessed in milk and 7 in serum. Perflutrrooctane sulfonic acid (PFOS). perf6uorooctanoic acid (PFOA). perfluorottonanoic acid (PFNA), and per0uorohexane sulfonic acid (PFHxs) were found i n nearly 10Y%of theser'um samples. PFOS and PFf1Awere found at the highest concentrations.PFCs were belowthe limitof detection in most milk samplre. Serum concentrations of PFDS. MA and PFHxS were lower(p t0.01) at the second visit compared to the first visit Living in North Carolina 10years or longer was related to elevated PFOS, PFOA and PFNA (p :s 0.03). These pilot data support the need to further explore perinatal PFCexposuresand potentially related health effects, as planned in the upcoming National Children's Study which provided the framework for this investigation. 02009 Published by Elsevier Inc. 2. t. Introduction and older demonstrated widespread exposure to perfluorooctane 3s sulfonic acid (PFOS) . perfluoroocranoic acid (PF4A), and perftuo- as zs Polyfluoroalkylchemicals (PFCs)comprise alarge groupofman- rononanoic acid (PfNA) during the last decade [20,2T 1 . 37 ze made fluorinated organic compounds used in numerous consumer Exposures of lactating women and young children to PFCs have 38 zr products and industrial applications such as food packaging mate- not been frequently studied, although a number of animal and 59 2a rial, non-stick cookware, protective coatings for textiles, carpets, recent human studies have suggested transfer to breast milk and .o 29, and paper surface car coatings or treatments, as well as in sur- across the placental barrier (22-26j. Developmental and repro- .+ 3o factants for commercial and industrial applications 11 ] . FFCs, and ductive health effects in animals, including reduced birth weight +s s+ more specifically perfiuoroalkyl adds (PFAAs), have been detected and gestational length, developmental delays and structural defects 43 az in wildlife, fish used for human consumption, and sera of humans especially in relation to PFOA and PFOS exposure have increasingly aa 33 in many different geographical areas worldwide [2-19] . Nation- raised concerns, although the developmental toxicity in Laboratory AS a, ally representative US sera biomonitor'ing data in subjects 12 years animals wasshown atdoses 100-500times ofthose seen inhuman 46 sera [2.27-291. Some expowre assessments in cord blood suggested 47 that PFAAs can also cross the placental barrier in humans ]3R,3t ]. +u Apelberg et al . [23] recently reported average cord blood concen- .9 Abbreviations: Cl, confidence interval;'IQR. interquaftile range ; LOO, limit of trations of 4.9 ng/ml (PFQS) and 1 .6ng/ml (PFOA) (n=299), while so detection . LOO, limit of quantificatlon;:Pfaas, periluomalky] acids: PFOSA, perflu- Spliethoff et al., reported the detection of PFAAs in new born blood s, orooctane sulfonamide: Et-PFOSA-Ac0li.2-(N-ethyl-perlluotnoctane solfonansido) spots confirming rhe transfer of PFAAs in urero 1321 . $x acetic add ; Me-PPM-AcqN. 2-(N-methyl-perlluoroocrane sulfonamido) acetic acid,. PFHxS, perfluorohexane sulforiic acid; PFOS, perfluorooctane su,fottic acid ; In two recent epidemiological studies . PFAA cord blood concen- ss PWA, perfluorooctannic acid ; PFNA, perfiuorononanoic acid; PFC. polyffuoroatkyl trations were related to anthropontetric indicators of fetal growth 54 chemicals-, VVTC, World Trade Center. at birth. and maternal pregnancy serum PFAA concentrations were ss Q2 ' Corresponding author at: UCLA School of Public Health, PO Box 1772, Los Ange- associated with child birth weight (22,241 . Based on the Danish 56 les. CA 9D095-1772, United States. Tel., ~t 310 206 5324; fax : +1 310 794 1805. National Birth Cohort, inverse associations were reported between s> E-moll address: ovehren(ducla.edu (OS. van Ehrenstsin). t Current address : US Environmental ProtectionAgency, Naitonat Center for Expo- gestational PFOA exposure and birth weight while no effects were sweAnalysis, Environmental MediaAssessrnetn Group.Mail code B243-O], Research reported for markers of fetal growth at birth, or postnatal develop- u Triangle Park. NC27711. United Stares. mental milestones [24,33] . so 089Q-6238jS -see front rnattet 0 2009 Published by Fasevier Inc. daia0,t078jJ .reprotox .2DD3.D3.DOl fMS`.23~'~JW__r-~~~ti_~I ~1F)sf~.16~i.~ied~ Rw~:..'^ti.; ~~.:-~r: -- p. 38 .=t~ M ~`. 2 o.S van Phrettstein er ol./Reproductive Toxicalogxxx (2009)xxx-xxx 61 Data on human milk PFC concentrations are still sparse. The Table 7 sz availabiedatabased onsmalisamplesizesfromChina(34),Sweden Limits of quantiftcation (tAQ)in milk andlimits ofdetaton (LOD) in serum(rigirrill. 63 (35j,GermanyandHungary(3fi],suggested detectable levels ofpre- Paty!fiuoroalkyl .i.-,1leniteals ' Milk LOO, Serum LOn e4 dominattriyPFpAandPFOS .Theconcentrations ofPFOS(131pgJml) 2-(N-ethy)-perOuoTaoctane , 0.60 0.20 es and PFOA (43.8 pgJm1) in 45 milk samples collected in 2004 from sulloriamido) awtic acid 66 women aged 22-43 years residing in Massachusetts have been 2.{N=methyl-periluoraoctane , O,SO 020 67 reported recently (251. Studies investigating the partition of PFCs sulfoaimido)acetic acid 66 into milk are largely lacking. One earlier study in Sweden (r1=12) Perfluott,butarte sultonate 0,30 ss suggested transfer of only about 1 Z of PFC concentration in serum oJ* 030- "0.10 ro into milk (35]. Temporal concentration changes in serum or milk of bso ~ .-,a.io . . - rt lactating women are unknown, as no study has assessed concen- Prrt7uomoctanesiifc~itt d. e:(PFl35A7 0.15 US: 7z trations in the same woman at two time points during lactation. PerOuoiooctenesulfornate(['Fq5), : . .' . '`0.60 ~ 'O.OS ra To evaluate infant and maternal exposure to PFCs and to a Perfluoroacia~naate (Pt~A) . . . . . . 0,30 0.10 T< range of other environmental components, as well as to compare ' Denotes not measured in serum. 7s concentrations across biological fluids (37j. the US Environmen7s tal Protection Agency (US EPA) conducted a pilot study entitled n Methods Advancement for Milk Analysis (MAMA~ This pilot study 2-4. Analysis ofmilk andserom for pFL's e26 79 was carried out to develop reliable collection and analysis methods 7s for the National Children's Study. including 100,000 children from In serum and milk, we determined the concentrations of PM PFOA, PFNA, rza ao pre-conception to age 21138]. We previously reported the MAMA 14:HxS, per0uorooCtane sulfonamide {PrOSAx 2-(N-methyl-perfluorooctane sutfonamido)acetic acid (Me-PPDSA-AcOH), Z-(N-ethyt-perfluorootcane sulfonamido) rso 131 at findings regarding phthalates {37] and the biological components acetic add (Er-PIFOSA-AcOH): perfluorobutane sulfonic acid and per0uorodecanok ,3z az of human trtilk ]38(, add were only measured in milk. The analytical method involved automated ,33 solid-phase extraction (SPE) coupled to remrsed-phase high performance liquid ,ai 2. Materials and methods chromatography (HPLC)-tandem mass spectrometry (MSJMS). Samples were run raa in sittglets and were re-analyeed only if the waterandJor matrix blanks were above la6 a4 2.1 . Studydesign and population 3 x limit of detection (L4D)_ The analytical procedures involving the use of stan- rsT dards, quality control, and bianks, as well as automated sample extraction were s3a conducted as published previously i43-461. The samples from both visits were ana- *oy as The design. of [he EPA MAMA study and basic methods has been described in lyzed together in March 2006 (serum) and November 2006 (milk). tao ss detail previously (391. In brief, 34 healthy, ErtgrGslrspeaking breastfeeding women For milk, sample preparation was conducted using automated off-line SPE (431. er between 18 and 38 years of age were recruited via newspaper advertisements, uni- One-ml of milk, to which we added 3 mi aro,i M formic acid and 50 pJ of internal sz aa versityemailpublications.andfliersdistributedtoctiniciansspecializ nginwomen-s standard solutiun, was vortex-mixed and sonicated, and placed on a Zymark Rapid m3 as healthorpediatricsbyanEPAcontractor(Westatlnc. .ChapelHill,NClThequesoon- Trace Station (2ymark Corp- Hopkinton. MA). PFCs from the milk were extracted on 144 0o rraire assessment and the collection of milk and serum specimens were aandutted an Oasis-HLB St'E column (waters Corporation, Milford . MA). The SPE eluate was ,45 s+ attheEPA`sHumanShttliesFacilityclinic(ChapelHill,NC)betweenDecember2004 evaporated at 55`C to -100p, under a stream o dry nitrogen (UHF grade) in a ,4s sz andJuly2005.wnmenwerebreastfeedingtheirfirst,secondorthirdchildandWere Zymark 7urbavap evaporator. and reconstitutedwith 300 p.1 orD iX formic acid.The u: 93 not required to exclusively breattfeed for participation in this study. The women reconstituted milk extract (-.400 14) was transferred to a polypropylene aurosam- 'All 94 donatedmi0candserurnsatnplesat2-7weeks(istvisit :n- IS milk;n+34serunty plerviaifnrtlteon-lineSPl:-HPLC-MS(I+ASanalysis .performed usingaSurveyorHPLC AA ss and at 3-4 months (2nd visie n - 20 milk ; n- 30 serum) postpartum. The partic- system (TherrnoFinnigan, SanJose. CA, USA). including one six-port switching valve ~so ve ipation of human subjects in the MAMA study was approved by the Institutional (Rtteodyne MX7960, Aohnerc Park. CA, USA) and one additional Surveyor LC pump, +s+ 57 Review Boards of the University of North Carolina's School of Medirine(IRB number coupled with a ThermoFinnigan TSQ Quantum Ultra triple-quadrupole mass spec- s52 lie 03-BPA-207)andrheCentetsfurDiseaseControlandPrevencion( BBnumber3$61). trometer equipped with a heated elecerospray ionization(HFSI) interface. The HPLC ,5g es The women participated in verbal and written informed consent prior [o admins- pump operated at a 300 W]jtnin flow rate with 20 mM ammonium acetate (pH 4) in sa soo trationofacorrtprehensivequestionnairewhichdidnotincludequestionsregarding water (mobile phase A) and aoetonitrile (mobile phase B). The extract was injected ss 1011 the offspring of study participants 139). into the liquid chromatograph system for ronKentration of the PFCs by on-line SPE on a Betasil C8 precolumn (3 mm x 10 mm, 5 AM: TherntoHypersit-Keysione, Beile- s57 102 2.2. Questionnaire fonte, PA, USA), chromatographic separation on a Berasil CS analytical HPLCcohtmn 's8 (2 .1 mm x 50 mm, 5 gm; ThermoFlypersil-Keystone), and detection and qwntifica- t59 ros A questionnaire regarding maternal residence, occupation, and dietary and tion by negative-ion HESI-MS(MS, e64 IN lifestyle factors was administered m participants at the first clinic visit. Ques- For serum, we used a modification of the on-line 5PE coupled to HPLC-MS(MS " ss +os tionnaire items were selecced to address potential routes of exposure to multiple approach described before 147]. Briefly, we added 250 p.l or 0.1 M formie acid and +as environmental chemicals (phthalares, phenols. PCBs, dioxins, PFCs . persistent 25p1 of internal standard solution to 100y,i of serum, and the spiked serum was rm 107 orginic pollutants, metals . and brominated flame retardantsy. The current analy- vortex-mixed and sonkated. The samples were placed on a Symbiosis on-line SPE i64 loa sis included the following questions that were thought to potentially relate to PFC system (Spark Holland, Plainsboro, NJ) for the precroncenrration of the anatyres on 16s log exposure routes : -Haw long have you lived in North Carolina?' (40-421 and "Does a Polaris C18 cartridge (7 Am, lOmm x l mm: Spark Holland). The analytes were 'fill r+a your home have an enclosed garage attached7.The latter question was selected as transferred onto a BerasilCB HPLCcoltm7n (3 mm x 50mm.5 gm; Thermofiypersil- let i some applications used in and around cars contain PFCs. e.g., external and internal Keystane, Bellefonte, PA). separated by HPLC (mobile phase A: 20mM amnionit:rn S66 z surface car coatings or treatments. acetate in water, pH 4: mobile phase B : methanoly, and de[ecred by uegative-ion "69 Tnttrotonspray-MS(MS onanAP14000 mass spettrometer(Applied Biosys[ems . Fos- 17(1 1+a 2,3. Sample collection andpreparlition terCity, CA). Reportablebreast Milk PFC crxtcentranons can fall below, the LOUdue to Ill concentration factors that are part tsf the extraction protocol.Thus limit of quantifi- +7a cation(LOQ)(3aL00) is used for milk samples and LOD is used forall other biological ,73 e14 The women were asked to fast for 1.sh before sample collection. The MAMA media, where sample concentration is not required. The t.OD in serum and the LOQ III ,is sample collection procedures for serumaand milk were published previously (39] . In milk are shown in Table 1 . ,7s ns Sampling details, including time ofday (between SAM and 2 PM) and the amount 117 of bodily fluid collected, were recorded in.ilte collection log. Milk (90ml or -3 oz) +1a wasexpressedIntheEPAcIinicusinga~flornmerCiallyava(lableeleetricbreastpUmp 2.5. Bfologfcatmarker rrnnlysia ,76 ns (Medela,McHenry,ll).MilkwaspumpedintoPFC-fteebottlesanddividedinto3ml szo aliquots in PF'C-free polypropylene-tubes. women's blood samples (about 20m1), Selected biologics in milk and serum were analyzed fareachwomai accordingto 177 tizr were collected into oan-beparinized glass. vacutainer tubes (Becton Dickinson, LabCorp's standard operating procedures for these assays as previously reported in 179 rzi Franklin Lakes, NJ) by an EPA nurse via venipuncture. After 1 h at room tempera- detail 139) . The assessed endpoints were in milk : Secretary imrnunogfobulin A,pro- 17 i2a turetpallowforclotting.b'Ioodsampleswerespunat3000rpmfor15minatroom lactin, tissue necrosis factor-at (TNF-a). interleukin-6 (IL-6). triglycerides, glucose . so 124 temperetumandserumwascollected.Allsamplesweresroredat-20-Candshipped and es[radlol; and in serum : prolactin, immunoglobins, TNF-a. IL-6, triglycerides, lei tzs ondryicetotheCDCsDivisionofLaborararySciences,NationalCenterforEnviron- glucose. and estradiol. in this investigation. the milkand serum concentration afthe rez Vs mentat Health (Atlanta. GA) for analysis. At the CDC. all samples were stored at or biological markers were used to explore possible relationships With the detectable las 127 below-20"C until analyzed. PFcs, ,~ p. 39 OS. Yon 9hrenstein er al./ Reproductive 7bxicologyxat (2009 )aoot-xnt 3 Table 2 those analytesfotwhich thefrequency ofdetecrion(>LOD)was>60% at both visits. 190 Q,S Percentage (number) ofserum and milk samples with PFCsA > LOD at visit 7 (serum Farrhosewomenwhodonated2serumsamples.thetnediandifferenceberameathe ,e, n-34 : milk n= i8) and visit 2 (serum n-30 ; rnitk a=20). concentrations for the same PFC at visit 1 and visit 2 was calculated and assessed 192 with the Wrlcoxon signed-rank test (non-parametricj.Spearman correlation coeffi- sya cienrs and related p values were calculated for correlations between the PFCs atvisit +e4 1 and visit 2, and between the PFCs and the biological markers in milk and serum . ws fiefationsbetweenoprioriselectedvariablesassessedbyquestionnatreandthePFCs =ae were evaluated using wilcoxon scores (rank sums) test- 11W cut-off points for cat- +s7 eggrizing selected variables were dec3ded a priori based on assumptions according +ae to data previously reported (40-42), and to aehieve approximately equal distribu- Ioo tion of numbers of subjects. across categories. Two-sided p values are reported. Alt 200 analyses were conducted with SAS version 9 (SAS Institute. Cary, NC). 20+ 3. Results aoa The median age of the women in this study was 31 .3 years zoa PFOSA Visit 1 'vtstt2 Me-PFOSA-AcOli = visita :visltZ_ : . _ (interquarile range (IQR): 271-34 .2 years), and the children's 2W median ages were 5.5 weeks (IQR : 4-6 weeks) at visit i and 13 2os weeks (t3-14 weeks) at visit 2. Three o the anatytes, PFHxS. PFOS aoe and PFOA, were detected in 't00X of women's serum samples at =r both visits, PFNA was detectable in 97% at visit 1 and in 100% of women's sainples at visit 2 (Table 2) . In contrast, in naik samples Zoe of just 4 women, only 3 of the analytes were >LOQ: Et-PFOSA- zto Act)H (1 .otigjt111) and Me-PFOSA-AcOH (0.7ngjml) were detected zr, in 1 woman at visit 1, and PFOSA was detected in 3 women at 212 the 2nd visit (0 .3, 0.5, and 0.6 ngjml). The remainder of the milk 213 samples from both collections were measured and found to have su b concentrations < tQQ. ~~5 b The distribution of PFC serum concentrations is shown in Table 3 . 216 Pertluorodeeanciate visit I b ~ visit 2 b Highest concentrations were found for PFOS with median values of 2,7 20.0 ngjml at the first visit and 16.9 ngjml at the second visit . PFS 7~U 0 concentrations were almost six-fold higher than the concentration 219 ' PFOS: pernuotooctane sulfonate; Et-PFOSA-AcOH: 2{N-ethy)-perfluorooctane oftheanalytewith thenext highestvalue,Pl:C)A,with tnedial7values ~za sulfnnamido) acetic acid ; Me-pFOSA-AcOF1 : 2-(N-merhyl-perfluorooctane sulfan- of 3 .5 and 2.9 ng/ml at the first and second visit, respectively. amido) acetic acid: PFHxS : perftuorahexane sulfonic acid ; P6LY5; perfluotnottanyl Median serum concentrations were significantly (p :50,01) rra suifanate ; PFDA: perfluorooctanoie acid ; PFNA: perfluorononanoic atid. lower for PFt)S, PFOAand PFHxS assessed at visit 2 compared to the m b Denotes nor measured in serum. concentration assessed at visit 1, based on samples of 30 women W4 who donated serum samples at both visits with the differences szs 1s Z.6 Statfsticul onalysis shown in Table 4. Accordingly, the concentrations of the detected 226 serum PFCs are reported for each visit (Table 3). Serum concentra- 227 us we calculated the percentage of detection for each analyre in serum.and milk. tiens of the same PFC were significantly correlated between the na 367 and derennined the median, range, mean, standard error, and selected percentiles. ,6a For values below the LOD .values equal to LOD/sqr2 were used 148,491. Furtturanal- two visits(Table 4). Due to the limited number of breast milk sam- 7213 +se yses. including relationships between visits and across media, were cor'iducted for ples with detectable PFC concentrations, we could not calculate the rao Table 3 Distribution (mean, standard error, medlan, seieued percentiles . IQB) of PFCs' in serum samples at visit i (n-34) and visit 2 (n-30) in ngjmi. Mean (SEM) 10th percentile , 25th percentile median 75th percetihle 90th percentile 95th percentile it<1R PFOS Visit,I : Visit 2 - PFOA visit 1 . visit z 21 .9 (7.9) . 18.8(1:5) . . . 3.99(035) 10(0.21) 11.7 9 .70 '9 .50 1.45 . - 13 .2 14 .0 . ' 2.20 2.40 2010 169 3 .50 . ' 2 .90 30,1 22 .6 4.64' 3 .70 37.6 30.2 6:0 4.65 45 .7 35 .5 8.70 5.0 16.9 8.60 2.40 1 .30 PFHas :.Visit 1 :'Visit 2 1 .94 (0 .27) 1 .50 (0.22)_ 2.40 . 3.40 3.80 1 .40 1 .70 - . . .' 2.90 4.60 1 .00 PFNA ' Visic.1 ; - Vislt 2 1.22 {0:12) 1.33 (0.09) 2.00 2.70 0 .90 790 2.40 0 .50 PFOSA'yisit i' Visit,2 O.W(0.01) 0.09(OS)7) <1.OD s(OD 0.10 0.10 0.10 047 0.10 0.15 0.20 0.07 Me-PFOSA-AeOH Visit f 0.23(0.02) KLOD <LOD 0,20 0.30 0.30 0 .40 0.16 visit 2 0.24(0.02) . <LOD . <LOD 0,17 0.30 0.40 0 .50 0.16 ' PFI)S : Pernuorooctane sulfonate; Et-PFOSA " ACOH : 2-(N-ethyl-perfluorooctane sulfonamido) acetic acid ; Me-PFOSA-AcOH : 2-(N-methyl " perfluoraoctane sulfonamido) acetic acid; PFtixS : perfluorobexane suifonic acid : PFY?S : perfluoraoctanyl sulfonate ; PFOA: perfluorooctanoic acid: FFNA : perfluorononanoic acid . Values measured cLOI? were impured by LODjsqr2. p. 40 4 ff,5. von Ehrerrsrein et ot I Reproduaive 7oxicvlogy xrnc (2009) xxx-xxx 'table 4 pifferenee and correlation in PFC serum concenirations(nWmi) between visit one and visit two. Table S Corretaions between concentration of PFC and inter]eukin-6 in serum at visit 1 (n .34) and 2 {n-30J. Pf(1$. '. . PFOA- . PFiixS PFNA -2-34 (-7.9. L0)-0.55 (-7.40, 0.0) . -0.40(-010.-010J 0.11 (-0,20,050) , c0.01 . . W,001 <0.001 0.10 atteTdtloih ~ . ' . ~D V311te -coeffldentv~ - - - -_ -- - 0.82 . --, - C0:001 0.82 <0.001 0.87 QI.001 0.71 <0001 ' Wilcowonsigned-ranktest (non-parametric)(n-30}. ' Spearman correlation coerfic(em IT and rela.ted p value Cn-30), Pros' visit 1 , Visit .Z . . PFOA visit i Visit 2 PF}ixS Correlation coefficient, a' .21 . . . .039-0 -0.'i5 O.p7 . p vaiiie 0,20 0.03 0,40 0,70 Viol 1 -0.11 OSO 231 partitioncoefficientfromserum tomilk,butcanconcludethatmiik Vistt'2 ', . . . 0,38 ' - 0,04 z~ concentrations were notably lower than serum concentrations. PFNA . rs Based on self-reported data, women had lived in North Carolina `visit 7 ~tl,[l03 . . . lA 234 for (mean, SF.M) 14 .6 (1-92) years. Interestingly, women who had V9sIt2 , -0,08 ' 0.70 zas reported living in North Carolina for 14 years or more compared ' Spearman correlation coefficient IV and related p value, Bolded values sigrdty z:s to those who had reported living in North Carolina less than 10 significant correlations. x+r years, had higher serum concentrations of PFNA. PFOA and PFOS zsa (p <i3.03) (Fig. 1). Furthermore, living in a house with an enclosed Serum concentrations of IL-6 were positively correlated with 244 aas garage attached as compared to living in a house with no enclosed PFOS (p =0.03) and PE7ixS (p=0_04) at the second visit (Table 5). x<s 240 garage attached, suggested a relation to higher concentrations of None of the other selected biological markers in serutn or in milk 2,6 241 PFIixS (ngJrnl; median, IQR, visit 1 : 2,2 (1.4) vs. 1 .1 (0 .6),p<0.401 ; showed a significant correlation with the PFCserum concentrations 247 242 visit 2:1.5(1 .4)vs.0.9(0J)p=o.tl3)andofPFOS(visit1 :25-4(16.9) ateither collection time paint.Tlterewasnosigniflcantrelationship ue 243 vs,14.4(9.9).p=0 .01 ;visit2 :21.2(11 .5) vs14.5(7.8)p=0.1) . between maternal age or parity and PFC serum concentrations in 249 our study (data not shown). Due to the small number3 and lack of ISO racial diversity in this pilot study based on convenience sampling xs1 (only. 3 women reported themselves as Black/African-American, zsz one as Asian and one as Hispanic), we could not analyze PFC con- zs3 centrations by ethnic group. 4. Discussion 255 In this pilot study of healthy lactating North Carolina women, 6 M of the 7 PFCs analyzed in serum were detectable at 2-7 weeks and 257 3-4 months postpartum . PFUS, PFOA, PFNA, and PFtfxS were found 25B in nearly 100% of the serum samples. PFOS, followed by PFOA and sss PFHxS were the compounds detected at the highest concentrations. 260 0 I -I - , , PFNA Visit 1 PFNA Visit 2 PFOA Visift 1 PFC by Visit (b) &0 F'FdA Visit 2 59 -E 40 Only a small proportion of milk samples had detectable values of 261 3 of the 9 PFCs analyzed in milk . Interestingly. serum levels were M_ lower for PFOS, PFOA, and PFHxS at the second visit compared to 263 the first visit, and prolonged time lived in North Carolina, as well as sw living inahome withenclosed garage attached,suggested arelation Us to elevated serum concentrations of certain PFCs in our sample; 266 however, these analyses were unadjusted and based on a small non- 267 random sample in this pilot study and thus should be considered sss exploratory. We can conclude that postnatal exposure to PFCs via z6s breast milk is likely to be low during the time period captured in 270 our investigation . z CD O 30 ti- 20 "d' 10 Data onPFCserum concentrations oflactatingwomen aresparse 272 and based on small sample sizes. Available data relevant for pre- a and postnatal exposures to PFCs are summarized In Table 6. Only n. one earlier study assessed both serum and milk levels, in 12 lac- 276 tating women in Sweden, and reported similar serum values to M oursforPFOS(med'san :18.7ngJml)andPFOA(3 .8ngfml)whiiecon- 277 centratiorts of PFHxS were higher (4.0 ngJml) in the Swedish study 276 1351. Based on data from the Danish National Birth Cohort, prena- z7a 0 tal matemaiserum concentrations appeared to be higher for PFOS re Visit 1 Visit 9 Visit 2 Visit 2 and PFOA in Denmark than seen postpartum in our study. Interest- zV ingly, in the Danish study, concentrations were lower in the second r s. Fig.] . Wand {b)Serum concentrationsofPFtvA,PFQA-andPFOS(ngiml)comparing than in the first trimester, possibly due to dilution of the PFCs with 2oe living inNorth Carolina :10 to <)O years at visit 1 and visit 2. Data are shown as box and whisker representations : open circles denare mean values with the medians blood volume expansion dueto pregnancy. but values in cord blood 254 denoted as a straighl line.'pe0.03 in Wilcoxon Scores (rank sums) tests for groups (n -50) confirmed fetal exposures (24.33] (Table 6}. The serum PFC 285, =t0years vs. K10years at visit I and vlsit 2 for each PFC. Numbers ofsubjeets in each concentrations seen in ourstudy comparewell with US serum data zas group: >i0 years : rr - 16. visit 1 : n- 15, Visit 2 ; 0 years: n-18, visit 1,n- 15, visit 2 . from NHANS 2003-2004, assessed in representative samples of 761 p. 41 O.S vpn EBrenatein et Qt. J Reprodncrive 7oxfcologyxxrc (2009)xxx-= 5 - Table C* Published data on average PFC concentrations in milk, maternal serum and cord blood . ..a 15~ pUF9jM&1tbc. '.. llrst;Einae :~n-3,4rssursed>t :'n~8 . .. 13"OP)PSIm! . - 1'FOA:93dS(3311PSIm4 ., . . . : ".' '-~'PFl:d:-14.5{i3,?)P81t?'d, . .~ . _::-PPNA :7.i6 (4ao).pehi+i. ~ ~rrr)pn, rrun, rrunun, rruoun,_ -PFHSall <LOD. " "!(c;couvestigntesastipliirg.at :.~ ., ; . : PFOS .(isiedian,range) : . . . .: : PFOS : 96% PFOA:89% . PFHxS- 51% PFrtA:'64z ' ': . :'PPHpA: frPDA. PF{laT1A PFDoDA, ms . :58% . .'-PFds:-100% _Aililkl'sault.ir-38(Leipzi8l---'- '~ : . . .. Mtmidi :113,:(28-239~.?j81L - ' " 3!~totEters aCpreterm inrattts, n=73 . - `: :Leipaigi 7i3:t33-~)aSJt: : . . . (Hsstigaiy)at.3-Y.weeks -. . 'PFOA:I6X. . . . , . 2lsousae, Cttina,Z004 . Swedtn,-individua! ritatched sera am: ~n: tdx.smialmkn(i2e0s0-.(4.:)t;s:spso;o-t2eodoaclo.m,-p;'.as:it. e. : , Hungary: 330 (96-539) rn81L 'PFOA, all: <LOD (<tA0-460) ng(L Milk :convenience sampling at 6ospitai volunteers, a,-19 Ranges}(ngj1 .) PPUS:4S-360; .PFOA:.47-210: mbis: 4-'t00 ; PPM: 6.3-62: .. PFDA,:.3-8='LS : PFlintl~k; 7b-56 . - . 1Ulilltart4-scrum :comreniqnce Milk (mean;SD)ng/mi:PFOS:- : s;ampIe .primipaFOUgvtO.Yneit : a~ 12 .. : 0201(0.F17) ; PFHxS: O ;Q85(0.047}: . : ~P1:0sA : o.a13 (0-oosr PPw+: NA; : ;4F~,~>'R?.4 MnDA ". ND :: -:*. ., :` ., ; . e`d'annnal caistposiietn'tik ' sa-mytes (n=25-90} - - Baltimore, MID, USA, 2004-2005 Date ofm11k colCection:3vieeks postpactum Cord blood, hospital based, stngteton deliveries (n,; 293) ' Serstni'(mean;SD)rtgltni:PFOS: 20.7 (70S);`FFHxS: 4.7 (29}: PFOSA: 024 (0.16); PP1JA: 0.80 (0-55); Pt1fTA: 3.8 (;.0). t1-53 . . (0.4i ):PFUnAA: 0.9D (033}' . . Compositemilk ngJrnl : PFOS UM (799~)4i23(Z004) ; PFHxS: 0-037 (i996)-0A16(20o4);PFOSR : <0.007 (1996}-<fl:oo7 j2004) : P . M 0-028 (1996)-0.020.(2004) : Won, <0.209 (1996)-02M (2D04) PFOA (median, range) : 1.6 (0:3-7.1) PFOS (median', range) ag/ndc 5.0 (~L0D(-02)-34.8) . Japan, 200.3 _ : . --- . . . Mate[na)"ptasma: ts6trimester . _ - -.2tidtri:testec.(-A.2.0. .0.).:' . . : : : . ... _ : Cord blood,)t -50 maternal plasma : 3rd trimester (n-15)cord bload(n-i5) ' , . . ., . . - '.Maternal ; :Isttrisitester. :PF05 PFOA: 5:6(7S)'. -Maternal,2ndtrimester.-PFOS; - :- z9.s (11:a):PFOA: -0.5 (1S) tord blood: PF06 :11.0 (4.7): PFOA: 3.7(3.4) . Matental, 3rd trimester serum rangea :PFOS(49-17.6ngfml). PFOA (<LOP to 2.3 ng/enl), PFOSA (<L0D to sLOD) Card bsood : PFpS {1.B-s3 ngjmi}, PFOA (<LUD to <t,OD). PFOSA (<J.ol) to -CLOD) ' PFC LOUs for serum. blood and mi)k varied in the different studies as reported in the original references. b No averages reported by authors. FM PFOA . PFHxSS, MM, PFDA. PPUnDA:10095 Milk : PFOS, PFHxS: 7F30k(n-12}* - PF0SA:67%(n-8},PFNA: is% {n-23:PFf1A:896(n-1) ' ' ' " --~ ..r :~. :. .~ .-~ . - . :~ . .: . .- . : ..,, . . . ., riim :. PFOS : FFH: :S, PF(}A;PFNA. PFDA,PFUnDA:100%(n-12) ; ` PFOSA : 75%-(11-91 . PFOS- 99% PFOA: i00X: Et-PFOSA--ACOH.Nie-PFO5A=AC0H. PFBiiS:PFHpA,:PFUA.PFQoA:i-40% -Maierstai, l-st trimester. PFCiS : lIIOX.PFOA 10t}x.(excepLn=i) . Maternal, 3rd trimester- PFOS: 100%,PFOA:20%,PFOSA :OS Card blood : PF05:100%, PFOA: OX, PFOSA :0% (n1 (341 (35) 122.231 (241 (3a1 zea females aged 12 and above, showing median concentrations for about halftheconcentration reported forcord blood from Denmark 3M zss PFOS and PFOA of 18.2 ngjmi _(1QR: :12 .4=27.3 ng/ml) and 3.6ngft1il 1241, Table 6. Wt ssu (1R : 2 .5-5.2 ng(m)), respectively (20,21) . Based on the NHANES A few investigations of PFCs in human milk have been con- 302 291 data, nation-wide serum concentrations dropped for PFOS, PFOA ducted in Sweden, China, Denmark and recently in the US (Table 6) aoa 292 and for PFHxS between 1999/2000 and 200312004 while those for (25,34-36} . Only one study assessed both serum and milk concen- 293 PFNA increased in the same time period 1201 . Our average levels trations and detected PFOS and PFHxS (n all 12 milk samples at s. are somewhat lower than repalted for females in the US in 1989 mean concentrations of 0.201 and 0.085ngtml respectively, selg- a6 295 {51 but sitnilarto other findings in samples collected between 1999 gesting partitioning of on average iX from serum to milk 1351. In W7 226 and 2005 [6,10,20,21,50j . In a recent US investigation, median cord the Chinese study, values of AFOS and PFOA in milk samples (rn =19) 20 blood levels forPFQS and PFOAof 5 and 1 .6 ngjml, respectively, were were in the range of 0.045-0.36and 0.047-0.21 ng/ml, respectively =a zsa reported (23j,This isabout athird toafourth (PFOS)and5(Y% (PFi7A) [34]. Milk concentrations are summarized in Table 6, supporting 310 299 ofthe concentrations we found in maternal serum samples, and also our findings of lower values in milk than in serum. as well as p. 42 O.S. Yon Ehrenstelp et at. / ReproductPve Toxfrolagy xu (2pD9)xrrrc-xxx asz suggesting regional differences in exposure levels [25,34-36j. the WTC fire or directly from the WTCs degradation) [59j further 3]B 3,3 PFAAs are strongly bound to the protein fraction of human blood supporting the notion of source related local variations of human 3rn 0,4 [10-51-53J . The protein concentration in human blood contains exposures to certain PFCs. Women who reported living in a home aan 3s main3yalburnin and fewer beta-lipoproteins and is about 3-5 times with anenclosed garageartached also had increased concentrations os higher than the protein fraction in human milk (casein and lactal- of PFlixS and PFpS in our sample . This may be due to certain mate- 382 a,? bumin). It has been shown that strongly protein-bound drugs are rials used in and around cars containing PFCs, such as post-market 383 a1a less likely to transfer to human milk than small non-ionic lipophilie applications of external and internal surface car coatings or treat- U< a+9 compounds [54[. This may explain why PFAA concentrations are ments. However, due to the small sample size in this pilot study, we 386 no much lower in human milk than in maternal serum,although irans- could not analyze the impacts of other variables, especially socio- aes 3x, fer of PFAAs to milk has been observed in animal studies, albeit at economic factors ; these findings are thus explorative and should be 322 much higher serum concentrations of PFAAs [26}. interpreted cautiously. aes az3 In our study, concentrations of PFOS, PFOA and PFHxS in serum The pro-inflammatory cytokine IL-6 was positively correlated to 3B9 324 were lower at the second visit compared to concentrations at the PFflS and PFHxS, respectively, at the second collection time point sss first visit. Since PFC concentrations measured in human sera have possibly indicating that certain PFAAs may be related to inflamma- a3s8,, azs half-lives ranging between 3A years for PFOA, 4.6 years for PFOS, tory processes : In line with these findings are recent results from 027 and 7.1 years for PFHxs [55j, these data suggest that processes experimental studies in mice, reporting suppression of immune 303 we related'to depurataon into breast milk might be occurring that responses following exposure to PFOS in urero [601. We did not see 304 sas we could not assess (possibly because we measured milk con- correlations with othera priori selected biological markers assessed Us centrations too late in lactation), or that there might be maternal in milk or serum, i:e., immunoglobulin, estradiot, prolactin orTNF ago 031 metabolic changes during lactation that may relate to this change a, Rodent studies 'using POAin concentrations orders of magnitude W 332 (i.e., changes in blood volume, body weight, or hepatic activities). higher than MAMA serum concentrations have shown a suppres- 348 333 Because PFCs are tightly bound to serum proteins, serum protein sion ofgeneticmarkers of inflammation after an acute exposure to a" 334 levels during lactation could have affected the concentrations of PFOA [fit [. Because our findings are explorative, future studies may 400 335 PFCs in serum. Unfortunately, we did not measure serum albumin want to address the role of chronic exposure to low dose PFCs in 401 as to test this hypothesis. Alternatively, if PFCs partition more into the inflammatory process. 402 337 liver than serum in the course of lactation, serum concentrations in this pilot study the number of women was relatively small and -D3 3:,e of PM could be affected as well.The possible transfer oPFCs to confines the investigation of associations, possible exposure path- 404 x+e milk may also vary at different times during lactation. The nature ways, and time trends after birth . In addition, the sample was not .as sw of the relationship between the suggested decline of PFC values in selected randomly, thus selection bias cannot be excluded. How- .ms 341 serum to concentrations in milk are yet unclear and insufficient ever, the participation of women was unlikely to be related to PFC 407 34 2 data exist to date to explain the relationship at this point. Few ear- exposures or to certain PFC exposure sources since they were most 446 30 lier reports suggested declines in breast milk during lactation for likely not aware of their PFC exposures. A further limitation of our 409 344 lipophflic compounds including dioxins. PCBs, and PBI)Es [56.57j. study is that we could not collect milk samples sooner after birth in 4yo s+s No other study to our knowledge, has investigated PFC concenira- view of ethical constraints in asking forthe colostrum milk_ Studies u+ as tion changes in serum or milk over time during lactation assessed in mice measuring PFOA concentration over the course of lactation 417 30 in the same women at two time points . However. it should be noted have shown that the peak in milk PFOA concentration occurs soon 4,3 W that our findings are ba sed on a relatively small number of avolun- after birth (Fenton et al., in this issue), a time that was not followed Q3 424 0.9 teer non-random sample of women and need replication in a larger in the MAMA collection scheme . Overall, the findings reported are 4+s aso study for further confirmation . Tao ei al . conducted a regression explorative and need further evaluation . .+6 3s= analysis of PFOS and PFOA concentrations in breast milk collected In conclusion. although infant exposure via breast milk is Iikely 417 asz at various time points from 25 different women within the first to be low, the cumulative daily infant intake of PFCs via breast milk s+a a% 6 months postpartum ; they concluded that values increased over per kg body weight could be appreciable for some populations or 4,9 3s. time of lactation [25j.However, since these findings were based on groups (Table S) . Since toxicological and pharmacokinetic data for 420 ass milk samples of different subjects rather than comparing changes PFC exposed infants are lacking, it is largely unknown if potential 42, 33s overtime in the same women. the differences may be due to intra- health effects in infants or during childhood may be related to cur- 422 3sT individual variation. rent exposure levels of PFCs. In urero exposure should continue to a2'J we Our investigation suggested that living in North Carolina for a be a concern as the MAMA serum PFAA concentrations are similar 42a 359 prolonged time period of 10 years and more was related to higher to values reported in two separate studies that have shown inverse 4as 3w serum concentrations of PFNA. PFOA, and P.FQS in our pilot study. associations between maternal serum or cord blood PFAA concen- no as+ However. further evaluation of this explorative finding is required . trations and infant birth weight [22,2AJ.Thus, the findings of this 427 3cr Poi nt sources may lead to elevated exposures as indicated by serum pilot study underscore the importance of biomonitoring maternal 428 353 concentrations ofPF(3A in persons living neara US facility using and and infant exposure to PFC as well as the need for further study .zo as4 producing this compound, that were notably higher than among of the potential human health effects of PFCs. In the upcoming US 430 3as the general US population [58J . A systematic surface water survey National Children's Study [38] PFC exposures in pregnant and lac- 431 ass conducted in North Carolina showed large variation in concentra- tating women and their children in North Carolina and across the a32 tiononasmallscaleindicatingaseries .of.sourceinputsaroundthe US will be further studied. 433 9se CapeFear Drainage Basin that may potentially result in pockets with a3sxao ianmcorenagseddoneoxrpsosautrtehse [640AJm.eCroimcpaanrRiendg CsreorsusmBlPoFoCd cBoannckenltorcaattiioonnss Conflict of interest 434 37s across the US showed highest concentrations for PF05 and second The authors declare that there are no conflicts of interest. 435 372 highest (or PFqA in Charlotte. North Carolina, in samples collected 473 in 2000-20011411, with a substantial decline observed in samples 474 collected in 2006 at the same locations [42j . Recently, elevated Acknowledgments +38 $xs plasma concentrations especially o FFOA . PFIJA, and PFHxS have ao been reported for personnel involved in the World Trade Cen- The research in this article has been reviewed by the National 437 a7r ter (BtrTC} disaster (i.e., from fire-fighting foams used to combat Health and Environmental Effects Research Laboratory, US Environ- 4" p. 43 ~"?`:~~=~ ._~ [i1Rp^4~~: ROME ~+ .. ~ .-~.f ^SXr.~.-AF.s .-. OS. vo Ehren.srefn er al./ReproducdveToxicofogyx=(2009)xxx-xxx 7 r 430 mental Protection Agency(EPA), andthe CentersforDisease Control 440 and Prevention (CDC) and approved for publication. Approval does 441 not signify this report reflects EPA or CDC policy. The findings in W this report are those of the authors and do not reflect the views of 40 the CDC. 711e use of trade names or commercial products does not 444 constitute endorsementor recommendation for use. e4s Thisworkwas supportedin part by the Intramural Research Pro- ass gram attheEuniceKertnedySttriverNational Institute ofChild Health 44Y and Human Development, National institutes of Health . Bethesda, 4aa MD. 44s Partial extramural funding was provided through the rec- 4so ommendation of the National Children's Study Intra-Agency 461 Coordinating Committee. 452 The authors would like to Richard Wang at the CDC for technical 453 assistance,Westat,inc .recruitingstaff(AndreaWare,BethanySrad- 45.t ford, Brian Karasek), and the US EPA nursing staff (Deb Levin, Mary 455 Ann Bassett, and Tracy MontillaX Finally we would like to thank the 456 MAMA participants. without whom none of this would have been 457 possible. 458 References 45s 400 aer 462 403 464 46s 46e 457 +sa aas 47o 47+ "72 rns 474 n7s 476 477 rre 479 480 4a+ 114 4ee 403 4s+ 485 48s 4e7 +as 490 490 41 492 493 4v4 4ss 4% aa7 4se 409 Sao s01 602 taa 604 Gals AM so7 Sos eoe sic ss i 512 613 st. 615 [ll 3M Company F]uorachemical use, distribution and release overview. 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Struggle for qualityindeterminationofperfiuorinatedcontaminantsinenvtronmentaland human samples. Environ Sci Technol 2006;40 :7854-60. [471 Kukletryik Z. Needham LL . Calafat AM. Measurement of IS perfluorinated organicacidsandamidesinhumansentmusingan-iinesolid-phaseextraction . Anal Cftem 2005;77.,6085-91. 1481 Hovnung RW. Reed l .D. Estimation of average concentraion in the presence of nondetectable values. Appl oecup Enviran Hyg 2008 :46-51. (491 CDC (Centers for Disease Control and Preventinn). General docutnentation an laboratory data. General information about the NHANES 2003-2004 laboratory methodology and public data files. Available : sIe s17 sre eis a2o e21 522 5xi xte 625 $zs s2l 528 629 soo 531 saz saa 5V s35 sas $37 su sae SO 54' 5e2 su saa s" s" s4r CAN ssv 560 5s+ ssz so asi ess sss $62 558 569 5ea sss 562 o6a 564 SO sce 667 556 esa sra 571 sre sTa 57" ors ste 677 $70 srs sea Sal ss2 583 554 sse so 6e7 ses 5GG syp 52, 592 593 sea sas 596 ser svs ssa 600 601 p. 44 8 O.S. von E#rrenstein er al. J Reproducrive 7oxkology :oot (2009)aorx-wrx Y eo2 http:J(www,cdc.govFnchsldatajnbanes]nhanes .03-04/lab_c.generaldoc .pdr; 1561 Hooper K Sbe J. Sharp M, et at. Depuration of poiybraminated diphenyl etllers e+4 603 2006 . (PBf7Es) and polychlorinated biphenyls (F'CHs) in breast milk from California an eo+ (SD] 01sen GW, Mair DC. Reagen WK .et aL Preliminary evidence ofa decline in per- first-time mothers (primiparae). Environ Health Perspect 2007:115!1271-5. 021 Ws Ftunraoctaaesulfanare(pFOS)andpertluorooctanaate(PFOA)concentrationsin {57] LaKindJS,BerlinCM,ParkCN,etaCMethodologyforcbaracterixingdistn'butions st2 No American Red Cross blood donors. Chemosphere 2007;68a05-11, eo : [511 Jones JD.Hu W. De Coen W. et al . Binding arperfluorinated fatty acids to serum ofincrementalbodyburdensof2,3,7.8-TCODandDAEfrombreastmilkinNOrtA sea American nursing Infants.JToxIcotEnvironHealth A2000;59:605-39 624 COB proteins. EvnvironTaxicol Chem 2003 :22 :2639-49. (58] Emmett EA, Shofet FS. 2hang H . et al . Community exposure to perltnoronc- 626 soe [52] Han X, Snow 7A. Kemper Met al . Binding of perfiuorotsctanoicacid ta rat and la noate : relationships between serum concentrations and expnsure snurces. J M ero human plasma proteins. Chem Res Taxico12009 :16:775-81, Otxup Environ Med 2006;46 :759-70. s2> oil 1531 8hattacharya AA. Grune T, Curry S. Crystallographic analysis reveals aomman ]59] Tao 1., Kanaan t( Aidous KM. et al. Biomonitoring of perRuorochemicals in sia e : modesof bindingofmedium and tong-chain fatty acids to human setvrn albu- plasma of New York State personnel responding to the World Trade Center ess e+a min,JMol8io12003 :303 :721-32. disaster. Environ SO Technot 2008:42:3472-8. wo as4 (541 Aschenbrenner DS. Veable SJ. Drug therapy in nursing . Lippinicort.Williams 8 (Sn] Keil DE . Mehlmann T. Sutterworth i, et al. Gestational exposure to perBuo- ss, 05 Wilkins ; 2008. rooctane sultanate suppresses immune function in B6C3F7 mice. Taxicol5ei 637 els 1551 Oisen GW: Bnrris JM, Ehresman DJ, et a1. Hatfdife of serum elimination of 2008 ;103 :77-85 . ssa s,? perfluorooctanesulfpnate, perfluorohexanesulfonate. and perBuorooctanoate E611 Guruge 1t5. Ytung LW. Yamanaka N, et al. Gene expression profiles in rat liver e3+ esa in retired fluorochernical production worker- Envifon Health Perspect treated with per0uoroortanoic acid (NFOA), Toxicol Sci 2005:89(1) :93-107. rae 2007;115:1293-305. p. 45 EL.,SEVIER Molecular and Cellular Endocrinology 304 (2009) 97-705 Coriteirts lists available at SciencefJirect .Moiec:ii~ar: and CelIulai~ Endo.arinology ,_ . . . . . . . . . . , . . . . . . . . . jourh;al.,hom:ep.age :;www :'e.lsevi er.:cornllot:atelm.ce . .. Phenotypic dichotomy following developmental exposure to perfluorooctanoic acid (PFOA) in female CD-1 mice : Low doses induce elevated serum leptin and insulin, and overweight in mid-life" Erin l? Hines a~*, Sally S. White b, Jason R Stankoa, Eugene A. Gibbs-Floumoy c, Christopher Laua, Suzanne E. Fentona ' Reproductive Toxicology Division, Office ofResearch and Developmen, National Health andPnvironmental iffects Research Laboratory, U,S Environmental Protection Agency, Research Triangle Park, NC27717, United States e Curriculum in Toxicology, i1NCChapel Hill, Chapel Hill, NC 2759% United States I Biological and Biomedical Sciences ?rogram/lnitiaCive forMaximfaingStudent Diversity. UNCChopel Hilt Chapel Hilt NC 27599, UnitedStates APT I C 6 E I N F O Article history: Received 27 January 2009 Accepted 24 February 2009 Keywords: PFOA Overweight Leptin Developmental Obesity Ovariectomy exposure ABSTRACT Thesynthetic surfactant,perttuarooctanoic acid (PFf)A) is a proven developmental toxicant in mice,eausing pregnancytoss, increased neonatal mortality, delayed eyeopening, and abnormal mammarygland growth in animalsexposed during fetal life . PFOA is found in thesera and tissuesorwildlife and humans throughout the world, but is especially high in the sera of children compared to adults. These studies in CD-1 mice aim to determine the latent health effects of PFQA following : (1) an in utero exposure, (2) an in utero exposure followed by ovariectomy (ovx), or (3) exposure as an adult. Mice were exposed to 0, 0.07, 0.1, 0.3,1, 3, or 5mg PFOAjkg BW for 17 days of pregnancy or as young adults . Body weight was reduced in the highest doses on postnatal day (PPIR) 1 and at weaning. However.thelowest exposures (0.41-0.3mgrkg) significantly increased body weight, and serum insulin and leptin (0A1-0.1 mg/kg) in mid-life after developmental exposure. PFOA exposure combined with ovxcaused no additional increase in mid-life body weight. At 18 months ofage, the effects of in utero PFOA exposure on body weight were no longer detected. White adipose tissue and spleen weights were decreased at high doses of PFOA in intact developmentally exposedmice, andspleen weight was reduced in PFOA-exposed ovx mice . Brown adipose tissue weight was significantly increased in both ovx and intact mice at high PFOA doses. Liver weight was unaffected in late life by theseexposure paradigms. Finally.therewasno effect ofaduhexposure to PFOA on body weight. These studies demonstrate an important window of exposure far low-dose effects ofPFOA on body weight gain, as well as leptin and insulin concentrations in mid-life, at a lowest observed effect level of 0.01 mgPFOA/kg BW. The mode of acrion of these effects and its relevance to humanhealth remain to be explored. Published by Etsevier Ireland Ltd. A: bbreviations ANOVA, analysis ofvariance; BMI, body mass index; BW, body weight ; C8, eight-carbon ; CV. coefficient of variation; DES, dietylstflbestrot; E-'. estradiol ; CD, gestational day ; t1R, Ha If-life ; IACUC. Institutional Animal Careand Use Committee; LH .luteinizing hormone; t.OD.limit afdetection ; LOtZ limit ofquantitatlon; NHANFS, National Health and Nutrition Examination Survey: NMR, nuclear magnetic resonance; NOAEt, no observable adverse effect level; ovx, ovariectomized ; PFAA,perRuoroaiky) add; PFOA, perfluorooctanoic acid: PFO5. perfluorooctane sultanate; PND. postnatal day ; PPAR. peroxisome proliferamr-activated receptors; SMR, standardized mortality ratio. * Disclaimer., The information in this document has been funded by the US. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory 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. Current address : US . Environmental Protection Agency, National Center for Exposure Analysis, Environmental Media Assessment Group. Research Triangle Park, NC 27711. United States Tel. : +1 919 541 4204-,fax : +1919 5412995 . E-mail address: hines.erin@epa.gov(EP Hines). 0303-7207jS - see front matter. Published by Elsevier Irel2nd Ltd. dai: 7010761j.mce.2009.02.027 p. 46 98 ER liines et of f Motecvtor ottd Cellufar Endocrinology 304W09)97-705 -1. Introduction Perfluorooctanoic acid (PFOA), one of the eight carbon (C8) perfluoroalkyl acids (PFAAs), is a synthetic, stable, persistent organic fluorine surfactant, used to impart water and grease resistance o various consumer products including non-stick pans, as surface treatments for clothing and food wrappers, insulation and fire-fighting foams. PFOA's high energy carbon-fluorine bonds are resistant to hydrolysis, photafysis and metabolism and thus itbioaccumulates and persists within bfota and environmental matrices, including water and soil, from the Arctic to the South Pacific (Lau et al., 2007). This ubiquitous environmental contaminant has an estimated half-life (tt/y) in humans of3 .8 years (Olsen et al .. 2007) and is found in production workers' sera, as well as those of the general population. Bio-monitoringstudies show detectable levels ofPFOAin human populations. The National Health and Nutrition Examination Survey (NHANES) reported that mean serum PFOA concentrations are declining in the USA population, from 5_2 ng/ml in 1999-2000 to 3 .9 ng/ml,-isr 2003-2004 (Calafat et aL, 2007). Arnsberg, Germany, an area with known drinking water PFAA contamination, had reported PFOA mean serum levels in 2006 of 25ngfml vs . 4ng/ml in unaffected German provinces (Hi9lzer et al ., 20081 The highest known non-occupational PFOA exposure via drinking water exists in the Little Hocking drinking water district where U.S. residents (Ohio and West Virginia) have mean serum PFOA concentrations of 478 ngJml (Emmett et at .. 2006). Children rnay receive significant PFOA exposures via dietary and water intake. Mean serum PFAA concentrations (such as perfiuorohexane sulfonic acid) were reportedly higher in children than in adult/elderly populations (Olsen et at, 2004). In the Little Hocking water district, an area of high environmental PFOA exposure, children age two to five and the elderly had significantly increased PFOA serum levels when compared with other age groups (Emmett et al., 2006). Although a bio-monitoring study in japan found PFOA in maternal blood, but not umbilical cord blood at parturition (Inoue et al., 2004, limit ofquantitation [LOt)J 35 .2 ngfml), a recent US. study (Apelberg et al., 2007) of human cord blood from term pregnancies reported relatively low levels of PFOA (limit of detection [LOD) 0.2 ng/ml) and another C8 compound, periluoroocane sulfonate (PFOS~ Within the reported study concentrations, the authors found that cord blood PFqA concentrations were significantly negatively associated with birth weight. A subsequent larger Danish study also found a significant negative correlation between maternal plasma PFOA and birth weight (Fei et al ., 2007) . There have been no consistent adverse health effects associated with occupational exposure to PFOA, in fact, the studies to date are contradictory. In worker populations. serum cholesterol and triglycerides have been positively associated with PFOA exposure while high density lipoproteins have been negatively associated with PFOA (Olsen et al., 2001). Categorical division of workers by PFOA exposure levels showed that, although notsignificantlydifferent from the other categories, body mass index (BMl) was elevated in the highest PFOA category (>30 ppm and BMIs >28,1995 data); this trend was not seen in the 1993 data set (Olsen et al., 1998). A retrospective cohort mortality study (n >6000)of PFOA-exposed employees reported significantly elevated standardized mortality ratios (SMR) in males with diabetes mellitus when compared to trten residing in West Virginia (minus the PFOA manufacturing area), Ohio, Virginia, Kentucky, Indiana, Pennsylvania, Tennessee, or North Carolina ; the SMRfor PFOA workers was not significantly increased when compared to West Virginia alone or USA residents (DuPont, 2006). In Atnsberg, Germany, PFOA was found to have an inverse correlationwith SMI in adults (Holzeret al., 20p8), The tips for PFOA in men and women are similar (Harada et al., 2005).Unlike humans, gender differences in PFOA clearance exist in rats (Kudo and Kawashima, 2003 ; Vanden Heuvel et aL,1991). Mice are the preferred animal model for evaluating the effects of PFDA on the developing fetus as they do not exhibit gender-dependent rt12 differences (Lau et al, 2006). However, even in the rat model system where the female rat rapidly excretes the compound, PFOA readily crosses the placenta (Hinderliter et al, 2005) and PFAAs are present in rat milk after PFOA treatment (Hind erliter et al ., 2005). Mice prenatally exposed to doses of PFOA at ?1 mg/kgJday exhibit developmental toxicity including decreased litter size, neonatal death, delayed eye opening, growth deficits, stunted mammary gland development, and early onset male puberty (Lau et al ., 2006 ; White et al., 2007 ; Wolfet al ., 2007). At higher doses and following long-term adult exposure, cancer endpoints associated with PFOA exposure in ratsinclude Leydig cell adenomas, pancreatic ac4nar cell adenoma/carcinomas, mammary f[broadenomas, and liver tumors (Blegel et at, 2001 ; Sibinski, 1987). PFOA increased estradiol (Ez) levels in male rats and PFOA-induced rodent Leydig cell tumors are hypothesized to arise from increased estradiol levels from aromatase induction (Liu et a1.,1996; Biegel et al., 2001). The majority ofthe ongoing work in the PFOA field has focused on the health effects following developmental exposure to PFOA . This study focuses on adult latent health outcomes in female offspring after developmental (gestational days (GD) 1-17) vs. adult (at 8 weeks of age, for 17 days) exposure to PFOA . Ovariectomized siblings were utilized in our second study block to address the role of the ovarian hormones in PFOA exposure-related health effects, as luteinizing hormone M)-overexpressing mice (Kero et al., 2003) displayed several phenotypic effects resembling those in our preliminary studies with PFOA. These studies address the role of developmental exposure and ovarian hormones in adult health effects including circulating leptin and insulin concentratians, adult body weight, and tissue and body weights in old age. 2. Materialsand methods 2.1. Animals Timed-pregnant CD-1 mice (Charles River Laboratories. Raleigh, NQ arrived on gestational day (GD)0 (sperm positive) at the US EPA where they were weighed upon arrival and randomly distributed among treatment groups. Pregnant dams were housed individually in polypropylene cages and received chow (tabDiet 5001, Plvtl Nutrition international t.t.C, Brentwood, MD) and tap water ad Nbftum. Two blocks or animals were rued in these studies. Stock 1 animals were dosed with vehicte (distilled water)r 7, 3, or SmgPFOA/kg body weight (EW) (n- 5.8.7. and 5 dams, respectively) ; block 2 animals were dosed with vehicle, 0,01, 0.1, 0.3, 1. or SmgPFDAft (n-I4 dams in all groups except SmgPFOA1k$ BW, which had 10 dams). PFCtA exposures are shown in the text as mgPFOA/kg. Animal facilities were maintained on a 12 :12-h light-dark cycle. at 2o-24 "C with 40-50g relative humidity. Animals were humanely treated as approved under National Health and Environmental Effects Research Laboratory protocols in accordance with the US EPA institutional Animal Care and Use Commhtee (IACCtC) . Sentinel mice, housed in the same room . were known to be free ofectofendoparasites and antibodies to certain viruses for the duration of these studies. 22 Dosingsoludim and procedures Pr-4A. as its ammonium salt (>98% pure), was acquired from Pluka Chemical (Steinhiem, Switzerland). PFC4 dosing solution was prepared fresh daily in deionized water. and the dosing solution was administered at a volume of 10 td/g. Mice received either water vehicle or PFQA at 0.01. o.t, 03,1, 3, or 5 mgjkg Sw by oral gavageonce daily overthe dosingperiods. The highest dose (5 mg PFnAjkg(day) was chosen because it wasknown to result in slightly reduced neonatal body weight gain with minimal postnatal mortality(tau et al ., 2006). 2.3. Experimental design 2.3.7, Developmental exposurelintact Titned-pregaant CD-1 tnice in=7-22 dams per dose group over two blocks) received 0, 0.01, 0.3, 0.3,1, 3, or 5 mg/kg PFOA by oral gavage on the mornings of Go 1-77. Dams were weighed daily prior to dosing and throughout gestation . At birth. pups were individually weighed and sexed. Pups within a treatment group were pooled and randomly redistributed among the dams of their respective treatment groups, and litters were equalized to 10 pups (both genders representedk Dams p. 47 EP. Hfnes et aL /Molecufarand Cellular Endocrinology 304 (2009) 97-705 99 or PFOA Developmtotal Dosing GO T-i7 3wks PFOA Adult t)4ainp (i wks o- I I G~ Birth ~e r wean & ovx 167B VAX Gluconse tolerance test 1Youn$) 2tSiwks _I MandRwar bleeds 42wka 7074w1is . . --- . .- GOO/ Mass Cotnposltlon Measured Glucose tolerance Test (aw Food Intake Monitoring I Is Mont" 1552 "MIlurt , . _. . . ... ._ ...... ..ullelt,lht monitored (developmentallyexposed, omt&Inracq.... ...,.. ._ ...... .. ... Fig. 1. Data collection schematic for study of developmentally and adult PFOA-exposed female mice. that delivered small litters (n <4 pups) were excluded from the remainder of the study_ Pups were weaned at 3 weeks of age al which point females were retained and housed 3-5 mfce per cage. Males were evaluated separately, at end pohtts that varied from those reported here. 2.3.2. Developmentolexposurejovariectwny A subset of developmentally exposed female siblings (OutgPFOAfkg, n-8 ; AO1rngPF0Akg, n-t5 ; O.1mgPF0Ajkg, n=11 ; 03mgPF0Ajkg, n+i4 : 1 mgPF4A/k& n -6; 5 mg PFOA/kg. n=7) wereovariectomized (ovx)at 2l or22 days of age, before the onset of puberty. Animals were sedated with keitamineJxylazine (87/T3 tngJkg IN respectively), their ovaries surgically removed through the abdomen, sutured, and animals were placed in warming cages until they regained alertness. 8uprenorphine analgesic EO-05 mgjkg) was given twice daily isn, for 48h in 03 ml volume for pain relleL 233. Adult expauue A separate cohort of mice received PFOA starting at 8 weeks of age. for 17 days (0 mg pFOAJkg~ n=s ;1 mg pFOA/kg. n =14; 5 mgPFOAf kg, n-14}. 23.4. Data collection The data collection scheme for these studies is shown in 5g.1 . Blood was col- lected from the submandibular veins of ovx and intact mice between the ages of 21 and 33 weeks. These bleeds took place between 74 :00 and 18:00. and 200 pp.l of blood (100 p.l ofserum) was collected for subsequent analyses ofinsulin and leptin. Females in all three exposure scenarios were weighedweekly up to 9 months ofage and then monthly until 18 months. The number of intact, developmentally exposed mice weighed weekiyjmonthly was 10. 25,20. 11, and 32 . respectively for 0.1A01. 0.1, 0.3, and 1.O mg PFoAjkg, If mice became moribund before the study ended. they were euthanized in compliance with the protocol approved by the US EPA IACtiC (early necropsy). Date and cause of early morbidity or mortality was recorded if known. At early necropsy (collected when necessary) or at 78 months. trunk blood, retraperitoneal abdominal white (found lying ventral to the intestines and reproducCtve tract) and interseapularbrown fat pads. abnormal growths, and organs were collected from all exposure groups- Relative organ weight is used to express organ weight as percent of total body weight. Data are reported here as mean :ESEM. 2.4 Glucose tolerance rest Glucose tolerance tests were performed on two groups of intact developmentally PFOA-exposed animals : old adults (17 months of age With 0, 0.1, 1 or 5rngPFOA]kg ; n-8-13 per dose group) and young adults (15-16 weeks old with o, 1 or 5 mg P60Ajkg, n-12 per dare group}. The night before the assay, fur was shaved from the lateral area of the lower leg to expose the saphenous vein and animals were fasted,The following morning,tbe mice wereweighed and blood glucose was measured by collecting a drug of blood from each mouse via puncture of the saphenous vein (or tail vein ifnecessary). The blood drop was placed on a test strip, and inserted into the calibrated glucometer (Accuchek Advantage) for baseline glucose measurement. The mice were then injected i.p, with n-glucose Solution (2 gft body weight from a stock solution) . and blood glucose concentrations were measured at 20.40, 60 and 120 (old mice) or 180 (young mice) minutes (t3-3min) after the Initial glucose injection . 2.5. Serum lepdn Serum (101e.1) collected by mandibuiar venipuncture was assayed for leptin by radio-immunoassay (l.inco Itesearch, St. CharIes.Mo) foNowing the manufacturer's protocol (a=5, controls ; n-19, 0.01 : n=16, 0.1,"n -11, 03 : n=24,1 mg PFOAIkg).The coefficient ofvariation (CV*) for the standards (concentration range of 0.2-20ngjml) ranged from O .l%to 8 .0%.The qualitycontrol standards termed QC1 (expected range 0.6-13 ogjml)snd QC2 (range 1.8-3 .8) had a measured concentration in these assays of0.9 and 29. respectively. 2.5. Senmt fttsuffn Sera (1Dld}colkcted by mandt']sular venipuncture were assayed for insulin by the ultra-sensitive single molecule Immunoassay by Singulex (Alameda, [A) following the manufacturer's protocol (n=9 control, n421, D.01 mgPFOAJkg; a- 16, 01 mgPFOAjkg: n=17. 03mgPFOAft: n .31, 1 mgPFOAJkg} Samples were analyud using a 384-well plate format with monoclonal capture and detection antibodies on the SingulexErrena equipment.TheCVs forthe assaystandards (range 19.5-5000 pgJtnl) were from 3% to 17% The assay LOD was 16pgJm1. All samples were run on the same day and the interassay CV was 9.4% and 5.1% far the 29 and 1745 pgJml quality assurance standards, respcrtively. 2T. Sodymasscnmpositfon whole body mass composition was measured in live, non-sedated 42-week-old mice using the Broker Minispec mil 7.5 LF50 Live Mouse Analyzer (The Woodlands, TX).'t]re minispec was a bencFttop 7.5 MHz time-domain nuclear magnetic resm nance (NMR)analyzer,which quantified body fat, lean tissue. and free body fluid in m[ce.The minispec was calibrated by Broker Optics, Inc. scaffprior to animal analysis with daily validations using Bruker standards. Mice were weighed and inserted into the instrument for analysis (1-2 min jan'snalj. Intact developmentally exposed female mice that underwent body mass composition analysis included contmL 0.01, 0-1, 0.3. and i mgPFOA1kg (n-e, 23, 20, 17, and 32, respectively) dose gmps. It was not possible to perform these measures with younger mice due to equipment availability. ' 2.8. Measurement af F2 in serum oJintact mice at 18 mondrs Serum Ez (25p3 volume) from 18-month-old mice (intact developmentally PFOA-exprsed animals) was measured with time resolved fluom-immunoassay (DELFIA Fstradiol I(it, WaBac oy, Finland) following the manufacturers recommendation using a VICTORxI) 1420 Multitabel counter, PerkinElmer Precisely time-resolved nuorometer(PerkinElmerl1ife&AnalyticalSciences,Shelton,Cf).The CVs for the standards (concentration range of 6.81-142Spgjmt) ranged from 02% to 4.Sg. 2.9. Feed consumption Feed consumption in 77-month-old, developmentally exposed . intact female mice (no 6 per dose group, 0, 0.1, 1 and 5 mgPFOAfkg} was measured in metabolic cages. Mice were allowed to acclimate to the cages for 1 week and food intake was monitored during the second week Mice were individually housed and provided with a pre-weighed amount ofpowdered lab chow ad ItbitmmThe remaining chow was measured at the end afthe week and the total amount was subtracted from the starting amount to determiae the total feed consumed for each mouse per week. 2.]0. Measuremenrofsertrm PFOA Trunk blood serum samples (-SOhI) from the femate CO-1 offspring at 18month necropsies or from mice tertninated at earlier intervals because of illness were transferred to the C1lC for PFOA measurement . serum PFOA determination was performed as described in Kukienyik et al. (2005) and White et al . (2009). 2.11. Statistics Data were analyzed using SAS 9.1(SAS Inc Cary, NC). Body weight on PNDI was evaluated as Utter means as these datawere obtained prior to mixing litteroffspring within a dose group, Bodyweights at each time point were analyzed with mixedetfects linearmodels (SAS Proc Mixed) to estimate means and standard errors and test for dose effects separately by timepoint .6oreach time paiqtthemodel included dose as a fixed effect p. 48 ' too E.p. Hines et aL /MOlettvlur and cellular Endocrinology 304 (2009) 97-10$ and cage nested within dose as a random effect Pairwise ttests were Calculated to test for any difference between each treatment group mean and the control group. Repeated measures analysis of body weightdata was evaluated two ways. First, weights were averaged by animal over eight 10-week intervals. This was done to decrease missingvalues in the data due to animal mortality In late life that was not equal across treatment, and to reduce the effect orlarge body weight variances later in life.This data smoothing method decreased uninformative short-term variations and also reduced the number of estimated parameters to a tractable value. A multivariate repeated measuresanalysis (5A5 Proc GLM) was performed on these reduced data. subsequent to a significant finding. comparisons were carried out as subtests of the overall analysis ofvarianee (ANOVA) at specific times ordoses. Second, sAS Proc Mixed was used to perform a univariate repeated measures analysisotthe weights across timeup until 37weeks (latestweightpoint atwhichno animals had died).The model estimated a separate fixed quadraticcurve aaosstime for each dose group and included a random effect for cage nested within dose . Correlation within animalswas modeled with a random effect foranimal nested within cage and dose in addition to an autoregressive covariance structure within each animal. In this way, the sovariance matrix For each animal's measurements included a constantcovarfance component at all time points in addition to a component wbkh decreased as time points grew farther apart. Tissue weight relative tissueweight, body composition, food consumption. and body weight measurements were analyzed using a one-way ANOVA (AYnnett's post hoc tests). with dose being the independent variable. A blocking variable was included to adjust for the group difference. No adjustment was made for multipre comparisons. Glucose tolerance was compared at Individual collection times by one-way t-test and over time by repeated measures and area under the curve comparisons according to the trapezoidal rule. Hormone (insurm. Ez and lep6n) concentrations were analyzed using ANOVA followed byYukeys post hoc test. Mortality data were analyzed with product limited survival estimates; tog-rank and wilcoxon tests were used to test for differences among the treatment groups in survival across time (SAS Proc t.ifetest). The revel ofsignificance for all tests was P < 0.05. 8 (B) 60 55 Sit 945 m 40 3 35 30 25 Q Q.Ol 0.1 03 ! Dose PFOA (mg/kg SW) 2 4 6 Period 5 8 3. Results 3 .3 . Developmental exposure 3.1 .7. Early and mid-life body weight effects There were no significant differences in live pup number at birth by dose group (p<0.05) and postnatal mortality was not addressed in this study as litters were equalized at birth. On postnatal day (PND) 1, the average weight of the developmentally exposed SmgPFOAJkg offspring was significantly less than controls (Fig. 2A); no other dose group demonstrated significant litter weight effects at PNDl At weaning, mean female body weights were still significantly decreased in the 5 mg PFOA/tg(13.9 g+ 0.8) compared to 18.4g+ 0.4 in controluntreated pups . At this time, the 1 rngPFOA/kg exposed animals were also significantly smaller than controls (p <0.05; 16.4 g* 0.3). Time-grouped mean body weights of the female offspring over their lifetime are shown in Fig. 2B. Beginning at 10-19 weeks of age, there was an increase in weight in the 0.1 and 0.3 rngPFOAft groups compared to controls ;by 20-29weeks ofage, femalesdevelopmentally exposed to PFOA showed significant dose-dependent increases in body weight at 0.01, 0.1, and 0.3mgPFOA/kg which extended to 40 weeks of age in the 0.01 and 0.1 mgPFOAJkg when compared with control (p<0.05). This is specifically shown at 20-29 weeks (Fig. 2C). where the0.01-0.3 mg PFOAfkg groups had average weights 11-15% higher than controls. Continuous analysis of repeated measures of body weight aver time demonstrated that the five dose groups were similar in intercept using a quadratic fit; however, the 0.01, 0.1 and 0.3 groups had a significantly greater week effect than control, indicating that their weights were changing at a more rapid rate than control or 1 mglkg. This is shown in Fig. 2D for weeks fi-37 (the latest weight collection time pointpriortodeath of anystudyanimalsl Additionally. the 0.1 mg/kg (p=0.056) and 0.3 mg/kg(p=0.046)groups had larger negative coefficients for weeicx (week squared), suggesting that their weights were starting to fall off more quickly at the later time points than the control groups (not shown). The estimated weight curve for the I mg PFOA/kgdose group was not significantly different from the control curve. Data from 5mgPFOAJkg exposed (D) $s so reo 45 3 aQ s mo 35 ~ 30 2s m Flg.2. Body weights of developmentally PFOAtxposed female offspring, Data are shown as mean *5M with _p <0,05 us. control . (A) Pup weight at PNDI after developmental PFOA exposure. (8) Body weight of female CD-1 mice over their lifetime, following developmental PFQAexposure over 8 periodsortlme period 1(0-9 weeks old). period 2 (1o-19weeks old). period 3 (20-29 weeks ord}.period4 (so-s9weeus otd),period 5 (AO-49weeksord). period 6 (50-59 weeksold), period 7 (6p-69 weeks aid), and period g (70-79weeks)) .{ C)Group mean body weights of female offspring at 20-29 weeks of age demonstrating excessive weight gain at tow doses. (D) Dosedependent quadratic regression fit to repeated measures of body weight in female mice. An increased rate ofweight gainwas seen in 0.Q7.0.1 . and 8.3 mgPFOAjkg dose groups compared to control and 'i mg PFOAjkg . p. 49 E.R Nines etaL /Molecular and teltutarE7idoainology 304 (2009) 97-T0S 101 (A) 70 .~] 60 so v 40 3 30 20 -{ 20 40 60 180 Time (minutes) iR s0 0 -t all 0 9.01 0.1 '11 0.3 1 Dose PFQA (mgAcg MV) Fig. 3 . Blood glucose concentrations following a glucose challenge after time 0 in (A) young (15-18 weeks ofd) and (B) old (70-74 weeks old) female CD-1 mice that were developmentally exposed to PFOA Data are shown as meanf SEM. mice, which were decreased in BW compared no control at PNDl, weaning, and 18 months, are not shown . 3.1 .2. Serum glucose tolerance testing Because of the excess weight gain in the PFOA developmen- tally exposed mice during mid-life, various tests were conducted on these animals (as close to the appropriate age as was possible) to examine the associated effects of these changes. No significant differences were detected in baseline glucose or serum glucose area under the curve in response to a glucose challenge irk young or old mice (control, 01, 1, or 5mgPFOAfkg. F<0.05. Fig. 3}. i1 a time-dependent comparison, youngmice exposedto 1 mg PFOAjkg showed a nearly significant increase in blood glucose over control animals at 20min post-glucose challenge (p=0.06). In old PFOAexposed mice, although there appeared to be dose-dependent glucose insensitivity at 20 rain, this shift in response was not significant. 3.1 .3. Serum insulin and leptin Serum insulin and leptin measurements were made using blood obtained via mandibular bleeds between 21 and 33 weeks (within the time frame of greatest observed body weight increases) using intact female mice dosed with 0, 0.01, 0.1 . 0.3, and 1 mgJkg PFOA . Insulin and leptin concentrations were significantly increased in mice developmentally exposed to the lowest doses of PFOA tested (0.01 and 0.1mgPFDA/kg). Although elevated from the control mean, leptin concentrations were not significantly different from control at 0.3 or 1 mg/kg PFOA (Fig. 4) . 3.t4. Fat to lean ratio At 42 weeks of age, mice from block 2 (control, 0.07, 0.1, 0.3, and 1 mg PFOAJkg) were evaluated using a Bruker Optics Body Mass Analyzer, which determines the amount of fat, lean and fluid Dose PitOA (mglkg) Fly, 4. Serum Ieptin (A) and insulin (8) in mice at 21-33 weeks of age ep <0.05 vs. control). Significant elevations are seen at 0.01 and 0.1 mgPFOA/kg. Data are shown as mean :LSEM. in live animals. There was no significant increase detected in % body fat-body weight in PFOA-exposed mice (data not shown). Developmentally exposed mice had no significant differences in fat:lean ratio across dose groups when compared to control (means ranged from 0.75% in controls to 0.9% in 0.01 and 0.1 mgPFOAjkg). Although no dose groups were significantly different from control, there was an increase above control levels of about 12% in mean % fat:body weight ratio and 14% in mean fat- lean ratio in the dose group exhibiting the largest change in body weight at 24 weeks (0.1 mgPFOA/kg). 3.1.4.7. Feed consumption. Feed consumption was measured in 17month-old, developmentally exposed intact mice (0, 0 .1, 1 and 5mgPFOAjkg) and no significant differences were found across dose groups when compared to controls (mean 26 gjweek consumed; individual data not shown). 37.5. We life organ and body weight effects A noted loss of animals after 36 weeks of age was further eval- uated (1=<g. 5}. At 51 weeks old, when there was no mortality in controls there were 20% 10%. 36%, and 6% mortality rates in 0.01, 0 .7, 0 .3, and 1 mgPFOA(kg groups, respectively. By 76 weeks, there was a 40% mortality rate in controls, and 32%,63%.60%, and 44% in 0 .01, 0.1, 0.3 and 1 mgPFAJkg groups, respectively. However, there were no significant differences between control and any treatment group at specific times in late life or in survival across time. Among those mice surviving to 18 months, body weight of PFOA-exposed females was no longer elevated compared to controls. Furthermore. a significant decrease in body weight at the 5mgPFDAjkg dose was noted (Table 1). At that time, ail remaining females were necropsied. Trunk blood, tissues (affected or of interest) and abnormal masses were collected, weighed and fixed for future study. Serum was collected and PFOA levels were measured . The majority of the samples across dose groups had PFOA concentrations lower than the limit of detection (0 .5 ng/ml) with detectable values at maximum concentrations of 3.5 ngJml, and 102 EP Hines et d( Molecular and Cellular Endocrenofogy 304 (2009) 97-705 Table t Mean or relative body and tissue weights at 18 months of age in intact and ovariectomized (ovx) female CD-1 mice . p. 50 nc. Denotes not collected from this dose group. a p <0.01 vs. rontrol. b p - 0.05-0.0'7. Relative weight (organ weightas percent of body weight}. there was no significant difference in serum PFOA concentrations across dose groups (data not shown). There were no significant differences in serum estradiol levels in developmentally exposed females at 18 months when compared to controls (non-cycling; mean range across doses from 12.9 to 15.8 pg/ml). Tissue weights from 18-month-old animals (intact and nvx) are shown in Table 1 . To determine if the weight o fat depots was altered in old animals due to developmental PFOA exposures, the retroperitoneal abdominal white and interscapular brown fat pads were collected and weighed. Abdominal white fat weight and relative white fat weight both showed significant decreases vs. control (p < 0 .05) at 1 and 5 mgPFOAJIcg. White fat weights were not collected for3 mlgjkg PFOA animals. At 18 months, interscapularbrown fat weight and relative brown fat weight both showed significant increases above control (p <0.05) at 1 and .3 mg PFOA/kg. The spleen was quite variable in weight among the different treatment groups, but there was a significant difference in spleen weight and relative spleen weight vs. control at 3 mg PFDArkg (p<0.05). Finally, at 18 months, no significant differences in liver weight or relative liver weight were detected. 3.7.6. Effect of ovariectomy on tissue and body weight gain A group of developmentally PFOA-exposed animals {0, 0.01, 0.1, 0.3, 1, and 5 mgPFOAJkg) were ovx at weaning and their body weight gain and adult health was assessed until they reached 18 months of age. At mid-life the weight of the control ovx females was expected to be greater than that of the sham-operated, intact controls (Fig. 6A; set ofbars at0 mgrkg), but the variance in the animal weights was appreciable and therefore the differences did not reach statistical significance . When comparing the body weights of animals in the ovx study by treatment group, over time (4 weeks to 18 months), using statistical methods consistent with those used for intact animals, there was no effect of PFOA (Fig. 6B) . Comparison of ovx animals to intact animals at 20-29 weeks, as shown in Fig. 6A. demonstrates an absence ofbody weight gain over control in the ovx animals treated with PFOA . PFOA exposure did not stimulate increased weight gain (above that of control ovx) at any developmental exposure level in the absence of the ovaries (also seen in Fig. 6B). The ovx animals were siblings to the intact animals in this study. The ovx animals were also assessed at 18 months . Developmentally PFOA-exposed ovx animals showed no significant differences in body weight when compared to control ovx females (control mean= 52.7 t 5.57 ; highest mean. 1 mgPFOA/kg=61 .5 :L 3.3 ; Table 1). rFon Dose (mgntg sw) Age Weeks Fig. S. Survival curves for developmentally PFOA-exposed female mice (0-t mgPFOAjkg). Although a fair number of PFDA-exposed animals die early, a tifetest (SAS) analysis detected no significant decrease in time todeath.The reasons for early life mortality are under investigation . Fig. 6. (A) PFOA-dependent cbanges in group mean body weight of intact and ovx female offspring at 20-29 weeks of age. There was no change in body weight or ovx animals; across PFOA exposures. (B) Dose-dependent quadratic regression fit to repeated measures of body weight in ovx female mice. Unlike intact siblings no significant differences were sem between dose groups in the ovx animals. p. 51 E.P nines et at. /Motecular and Cetfalar FndocrinotoV304 {2009)B7-r05 103 As with intact siblings, the tissue weights of ovx animals are reported in detail in Table 1 . In ovx animals. neither abdominal white fat pad weight, nor relative abdominal white fat pad weight, were significantly different from ovx or intact control levels . This varies slightly from intact siblings. where the white fat pad was significantly decreased in size; although, in animals that weighed significantly less than intact controls. Among PFOA exposed ovx animals, both interscapular brown fat weight and relative brown fat weight (data not shown) showed significant increases above control ovx levels at 1 mgPF0Afkg (p< 0.05); no other dose groups showed a significant increase. This is similar to the effect seen in intactanirnals, and was significant at the same dose. Spleen weight (data not shown) and relative spleen weight in ovx animals was highly variable at 18 months. and showed decreases, albeit not highly significant, at the 1 and 5mgPF0A(kg doses (p=0.06 and p=0.05, respectively ; Table 1). 1 and 3mgPFOA/kg (not 5 mgjcg) were the doses in the intact animals showing the largest decreases in relative spleen weight compared to controls. Finally, relative liver weight showed no signiftcantdifferences across dose groups when compared to ovx control. 3.1.7. Lack ofeffectsfram aduit P>:'OA exposure At 18 months of age. body and tissue weights were recorded in adult PFOA-exposed mice. Adult PFOA exposure had no effect on terminal body or organ weights . When a comparison of data from 18-month-old adult intact and developmentally exposed animals in the 0, 1 and SmgPFOAjkg dose groups was made, body weight, brown fat weight. and white fatweight of the 1 mgPF0AJkg developmentally exposed animals were significantly higher than the same dose in adult-exposed animals (data not shown). 4. Discussion These studies demonstrated the effects of developmental PFOA exposure on CD-1 female mousebody and organweight, as well as serum lept'rn and insulin in adulthood. lin the developmental PFOA studies, a dose-dependent dichotomy of phenotypes was present in intact female mice; latent effects present following high doses were notpresent in mice exposed to low-dose PFOA and vice versa. Although there was no detectable change in body weight neonatally. low-dose PFOA exposures (0.01. 0.7, or 0.3 mgPFOAjkg) led to significantly increased mean weight and rate of weight gain in mid-life (up to and including37 weeks of age) and acoincident significant elevation of serum leptin and insulin values between 21 and 33 weeks (0.01 and 0.1 mg PFOA/kg). Our low-dose hormone data indicate potentially important metabolic changes that mechanistically support the findings of increased weight in the lower dose groups. Previous dosimetry work in our lab has shown that in utero exposure to PFOA in the mouse translates into an extended developmental exposure period via iactational exposure (all of gestation and nearly 3 months postnatally; White et al.. 2009 ; Wolf et al., 2007 ; Fenton et al.. 2009). This long exposure may lead to reprogramming/metabolic events that govern fat metabolism or appetite control. Although we were unable to perform some of the other end points of interest during this time period of greatest weight gain . our findings relating leptin and insulin concentrations to the time ofoverweight in PFOAexposed mice support our theory. Other environmental chemicals, termed environmental obesogens (d'ietyistibestrol (DES). 20H-E2, 40H-E2, genestein and bisphenol A), have been shown to induce obesity in adulthood after low-dose developmental exposure.wbile inducing weight loss at higher doses (Grun et al ., 2005; Newbold et al ., 2005; Miyawaki et al ., 2007) and are reviewed further within this issue. Serum leptin was significantly elevated in mid-life in the lowdose PFOA-exposed groups. This effect occurred at the same PFOA dose range as overweight in these animals, congruent with a leptin-resistance mechanism ofaction foroverweight,as previously reported in humans (Considine et al., 1996). Others have reported increased leptin with developmental exposure to environmental obesogens including DES (1Vewbold et al.. 2007}. Low-dose (0.01 and 0.1 mg PFOA/kg) developmental PFOAexposure that led to increased serum Ieptin and body weight also increased insulin values at 21-33 weeks. This suggests that the insulin resistance mechanistic pathway could also be affected and play a role in developmental PFOA exposure-induced overweight in mice. In an insulin resistance scenario, there are raised plasma glucose levels (elevated, but not significant, at 15-16 weeks in our study), reflecting the loss of a post-challenge peak in insulin response (reviewed in AAontecucco et al., 2008). Insulin resistance is known to be associated with excess abdominalfat in normal and overweight women(Carey et a1,1996) . High plasma levelsofinsulin andglucose,dueto insulin resistance, are oftenassociated with type 11 d'sabetes and metabolic syndrome in humans, and thus this effect of low-dose PFOA developmental exposure and its association with increased serum insulin are important The ovx data were difficult to interpret. The lack of additional weight gain with developmental PFOA and avx may reflect a "ceiling effect" or that ovx-induced weight increases may have masked any effect ofPFOA . Alternatively. as weight gain and metabolic hormones can be regulated by estrogens. the role of the ovaries in developmental effects of PFOAwas explored by using ovx animals . The potential importance of the ovary in the effects of PFOA was based on the observation that LH-transgenic (overexpressing) mice (Kero et al., 2003) were phenotypically similar to ours (increased body weight, increased brown fat depots, and predominant ovarian cysts not discussed in this paper). We hypothesized that removal of the LH target (the ovary) in our study may reveal the mode of action for PFOAeffects for the increase in brown fat and possibly theexcessive weight gain . Ovx animals typically gain body weight in excess vs. intact animals (Kamei et al, 2005}. The critical role of the ovary in weight gain of intact PFOA-exposed females beyond that of ovx treatment-matched siblings in the 0.01 and 0.1 mg PFOAjkg groups was novel and signifies the ovarian axis as a potential mediator of PFOA-dependent mid-life weight changes. Another potential mediator of these intertwined low-dose PFOA-induced effects is the peroxisotne proliferator-activated receptor (PPAR) activation pathway. PPAR gamma (PPAR-~y) and PPARalpha (PPAR-at) are involved in lipid metabolism in adipocytes and liverJskeletal muscle, respectively (reviewed in Medina-Gomez et al_, 2007; Abbott, 2009~ These PPAR isoforms are known to irnluence lipogenesisjweight gain and have been shown to be regulared by environmental compounds such as tributyltin (Grun et al., . 2006 ; reviewed in this issue). Weight loss events in leptin-deficient, obese, and insulin-resistant mouse models have coincided with PPAR-regulated changes in gene expression (Holvoet, 2008). A down-regulation of PPAR isoforms involved in energy expenditure, lipogenesis or fatty acid synthesis have been reported in adipose and skeletal muscle of ovariectornized mice (Kamei et al., 2005). PFOA has been shown to be a PPAR activator in liver tissue (high doses) and cell lines, and to be required for PFOA-induced devel- opmental toxicity fn mice (Takacs and Abbott, 2007 ; Abbott et al 2007 ; Abbott, Z009). ]f PPAR activation via receptor binding is a primary mode of action for body weight effects following PFOA exposure. the decrease in the PPAR receptors following ovariectomy and decreased circulating estrogens may explain the lack of effect ofPFOA in ovx mice . However, PFOA-induced consequences of PPAR activation following a developmental exposure are just beginning to be evaluated. After 40 weeks of postnatal age, an increase in mortality was detected in alt animals. There are previous reports in the literature ofincreased mortalityin non-treated CD-1 mice,attributed primar- p. 52 104 EP. Hines et oL /Moiecuiar and CeNufar Endocrinology 304 (209)197-70 ily to thymic lymphomas (Son, 2004 ; Taddesse-Heath et al ., 2000), Because of this confounding circumstance. repeated measures of body weight were only followed out to 37 weeks of age. The other half of the phenotypic dichotomy caused by developmental PFUA exposure was also novel. Developmental exposure to higher doses of PFOA (1, 3 and 5 mg PFOAJkg) led to a vastly different phenotype from low-dose PFOA exposure. This effective PFOA dose dichotomy may manifest itself in our study via unique modes ofaction ; the animals with highest dose(s) ofdevelopmental PFOA exposure have decreased early life body weight and terminal body weight (5 Ing PFOAjkg) with significant decreases in white fat weight at 18 months (1 and 5mgPFOAjkg), significant increases in brown adipose (1 and 3 mgPFOA/kg~ and significant decreases in spleen weight (3 mgPFOA/kg) findings that are absent with the lower doses of PFOA. Others have reported dose-dependent loss of white tissue adiposity in adult male mice after PFOA exposure (0.02% PFOA weight/chow weight, which translated to approximately 32mgPFOAfkg BW daily) with fat loss, without fat cell number loss, that is PPARy-independent with fl-adrenergic activation (Xie et al, 2082). In that same study, investigators also reported white fat and body weight decrements at higher doses that were absent at lower doses. Yang et aL (2002) showed PFOA-dependent weight loss was abrogated in PPAR-a null mice, indicating that PPAR-a is a probable regulator of weight loss in the high dose animals. In subsequent studies, Me et al. (2003) showed that after cessation of exposure of adult male animals to PFOA (0.02% PFOA weight/chow weight, 32mgPFOA/kg BW) daily for 7 days followed by 10 days recovery, weight loss and white adipose levels returned to baseline, which confirms the importance of developmental exposures for the latent effects reported here. In our model with developmental PFOA exposure we see permanent weight loss and white adipose tissue loss at the high dose of PFOA . However, there may be merit in further exploring these mechanisms of action, as P-adrenergic receptor upregulation is also associated with increased brown fat mass in winter-acclimated animals (Feist,1983), and this tissue was associated with high dose (and not low dose) effects in both intact and ovx animals in this study. Although we suspected alleviation of effect in the brown fat pad by eliminating the ovary (based on phenotypes in Kero et al., 2003), significant increases in brown fat were seen at 1 mgPFOAjkg in both intact and ovx animals. At the 18-month time point, some endpoints remained unchanged across dose groups including live rsize. Earlier work has shown significant hepatornegaly after developmental PFOA exposure (1 and 3mgPFOA/kg) observed out to at least 3 weeks after birth (the latest time point evaluated; Wolf et al., 2007; White et al., 2007).Thetransient nature of hepatomegaly hasbeen illustrated in other acute adult exposure studies (reviewed by Lau et al ., 2007), and is further confirmed in these studies (intact and ovx). A final important component of these studies evaluated adult vs. developmental exposure to PFOA on body tissue weights. These data suggest that the timing of dosing (adult vs . developmental 17-day PFOA exposure) was critical for latent effects . There was no effect of 77-day adult PFOA exposure on any endpoint in this study(early life orlatent) when compared to age-matched, vehiclegavaged controls. In conclusion, the timing and dose of PFqA exposure for induction ofdichotornous, persistent, adult health effects in CD-1 female mice are critical . Developmental, low-dose PFOA exposure led to increased weight in adults, with increased serum insulin and leptin, a health effect not seen in high dose animals. No observable adverse effect levels (NOAEL) for body weight gain, serum Ieptin and insulin concentrations were not determined in,this study; bpt 0.01 mg PFOAJkg had a significant impact on these particularly sensirive end points. The ovary appeared to play an important role in the overweight effect in mid-life, and it is proposed that there is a common mode of action, potentially dysregulation of PPAR and its signaling through ovarian hormones, that may be responsible for these low-dose health effects. Further studies addressing longterm PFOA-induced health outcomes inmice should focusattention on internal dose relative to the low-dose health effects seen in this study, as well as the mechanisms of action, so that any relevance to human health effects can be addressed . Acknowledgements We would like to thank Btuker Optics, Inc. for the use of the Bruker Arlinispec mq 7.5 LF50 Live Mouse Analyzer and Harry Xie and Basil Desousa of Bruker Optics, Inc. for their technical assistance. We would like to acknowledge Antonia Calafat and her laboratory staff, Kayoko Kato and 7suzsanna Kuklenyik, in the Division of Laboratory Science, National Center for Environmental Health, Centers for Disease Control and Prevention for the analysis ofserum PFOA concentration from 18-month-old developmentally exposed female mice; Donald Doeriler, Experimental Toxicology Division, U.S. EPA, and Judy Schmid, Reproductive Toxicology Division (RTD), US. EPA fortheir statistical support : Deborah Best, RTD,forconducting the estradiol assays; Veronica Luzzi, David Gibson and staff at the Core Laboratory forCtinical Studies atWashington University in St. Louis, MO, for performing the serum insulin assays, and finally. 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