Pharmacokinetics of Perfluorooctanoate in Cynomolgus Monkeys

J. L. Butenhoff*,1, G. L. Kennedy, Jr{dagger}, P. M. Hinderliter{dagger}, P. H. Lieder*, R. Jung{ddagger}, K. J. Hansen*, G. S. Gorman§, P. E. Noker§ and P. J. Thomford

* 3M, St. Paul, Minnesota 55144; {dagger} DuPont Haskell Laboratory, Newark, Delaware 19714; {ddagger} Clariant, Sulzbach D-65840, Germany; § Southern Research Institute, Birmingham, Alabama 35205 and Covance, Madison, Wisconsin 53704

Received May 21, 2004; accepted September 7, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pharmacokinetics of perfluorooctanoate (PFOA) in cynomolgus monkeys were studied in a six-month oral capsule dosing study of ammonium perfluorooctanoate (APFO) and in a single-dose iv study. In the oral study, samples of serum, urine, and feces were collected every two weeks from monkeys given daily doses of either 0, 3, 10, or 20 mg APFO/kg. Steady-state was reached within four weeks in serum, urine, and feces. Serum PFOA followed first-order elimination kinetics after the last dose, with a half-life of approximately 20 days. Urine was the primary elimination route. Mean serum PFOA concentrations at steady state in the 3, 10, and 20 mg/kg-day dose groups, respectively, were 81, 99, and 156 µg/ml in serum; 53, 166, and 181 µg/ml in urine; and, 7, 28, and 50 µg/g in feces. Mean liver concentrations reached 16, 14, and 50 µg/g in the 3, 10, and 20 mg/kg groups, respectively. In the iv study, three monkeys per sex were given a single dose of 10 mg/kg potassium PFOA. Samples were collected through 123 days. The terminal half-life of PFOA in serum was 13.6, 13.7, and 35.3 days in the three male monkeys and 26.8, 29.3, and 41.7 days in the three females. Volume of distribution at steady state was 181 ± 12 and 198 ± 69 ml/kg for males and females, respectively. Based on the result of both the oral and iv studies, the elimination half-life is approximately 14–42 days, and urine is the primary route of excretion.

Key Words: perfluorooctanoate; PFOA; pharmacokinetics; monkey; APFO.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perfluorooctanoate (PFOA), the dissociated carboxylate anion of perfluorooctanoic acid and its salts, has been found widely distributed in sera from the general population of the U.S. (Olsen et al., 2003aGo,bGo, 2004aGo,bGo). As a stable, soluble surfactant, the primary commercial use of PFOA has been in the form of the ammonium salt (APFO) as a processing aid in the production of various fluoropolymers.

In rats, PFOA was rapidly absorbed following a single gavage administration and peak blood levels were attained 1–2 h after dosing (Kemper, 2003Go). Gibson and Johnson (1979)Go found that 93% of an oral dose given to male rats was absorbed within 24 h. PFOA is not known to be metabolized (Kuslikis et al., 1992Go; Ophaug and Singer, 1980Go; Vanden Heuvel et al., 1991Go) and has been demonstrated to undergo enterohepatic circulation in rats (Johnson et al., 1984Go). Excretion of PFOA occurs in both the urine and the feces (Johnson et al., 1984Go; Riker, 1980Go). Biliary excretion was slower in male than female rats (Kudo et al., 2001Go) and less than 1% was reported excreted in bile of either male or female rats (Vanden Heuvel et al., 1991Go).

The elimination rate of PFOA varies greatly among species, and, in some cases, between sexes within species (Butenhoff et al., 2004aGo; Kennedy et al., 2004Go; Kudo and Kawashima, 2003Go). Of the species studied, female rats eliminate PFOA most rapidly, with a half-life of hours compared to several days for male rats (Davis et al., 1991Go; Hanhijärvi et al., 1982Go; Kemper, 2003Go; Kojo et al., 1986Go; Kudo et al., 2001Go; Ohmori et al., 2003Go; Ophaug and Singer, 1980Go; Vanden Heuvel et al., 1991Go; Ylinen et al., 1989Go). Importantly, PFOA does not appear to accumulate in blood of female rats (Kemper, 2003Go). In dogs, Hanhijärvi (1988)Go found plasma half-lives of 473 and 541 h in two male dogs and 202 and 305 h in two female dogs following a single iv injection.

Differences between species in elimination kinetics do not appear to be related to differences in serum protein binding (Han et al., 2003Go; Kerstner-Wood et al., 2003Go), and are likely due to hormonally controlled transport processes (Davis et al., 1991Go; Hanhijärvi et al., 1982Go; Kudo et al., 2002Go; Ylinen et al., 1989Go). By comparing PFOA clearance rates and renal transcriptional expression of mRNAs for organic anion transporters (oat1, oat2, oat3, and oatp1) in castrated male rats, castrated males given testosterone, males given estradiol, ovariectomized female rats, ovariectomized females given estradiol, and females given testosterone with non-modified controls, Kudo et al. (2002)Go suggested that sex hormone modulated differential expression of organic anion transporters and organic anion transporting polypeptides in rat kidneys may explain the large differences observed between male and female rats in the clearance of PFOA. Specific transporters responsible for these differences have not been conclusively identified.

The human serum elimination half-life of PFOA appears to be on the order of one to several years based on studies of workers that have either retired of have been removed from workplace exposure (Burris et al., 2002Go; Ubel et al., 1980Go). The best current estimate of elimination half-life in humans from an interim study of retired workers is 4.4 ± 3.6 years (Burris et al., 2002Go). Known re-exposures were accounted for in the latter study, and the potential magnitude of continuing exposure from possible environmental sources was considered inconsequential.

The low elimination rate of PFOA in humans, together with evidence of widespread exposure of the general population, underscores the importance of understanding the potential hazards and pharmacokinetics of PFOA in order to characterize risk. The potential health effects of PFOA have been examined extensively and recently reviewed (Kennedy et al., 2004Go; USEPA, 2002Go). Butenhoff et al. (2004a)Go have recently published a risk characterization for general population exposure to PFOA that relies on comparisons of serum PFOA concentrations in the general population with those associated with endpoints from toxicological studies to estimate margins of exposure. An understanding of comparative pharmacokinetics is a key factor in this risk characterization methodology.

Monkeys, as non-human primates, are often used to develop pharmacokinetic data to assist in understanding potential pharmacokinetics in humans. To describe the pharmacokinetics of PFOA in cynomolgus monkeys, this article draws on analyses for PFOA in serum, urine, fecal, and liver samples collected during the course of the six-month oral study of APFO in male cynomolgus monkeys (Butenhoff et al., 2002Go) as well serum and urine samples taken after a single iv dose of K+ PFOA given to male and female cynomolgus monkeys. The pharmacokinetic measurements in the six-month oral dosing study were used to determine the steady-state concentrations in serum, urine, and feces in relation to varying dose and time. In addition, the elimination profile of PFOA was followed after cessation of dosing. The iv study was conducted to establish classical pharmacokinetic parameters in male and female cynomolgus monkeys.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biological Samples: Six-Month Oral Toxicity Study
The biological samples (serum, urine, feces, and liver) were collected from cynomolgus monkeys during and following six months of daily oral dosing with APFO. The methodology, results, and conclusion of the toxicologic endpoints evaluated have been published (Butenhoff et al., 2002Go). Briefly, groups of four to six male monkeys each were given daily (seven days per week) oral doses (by capsule) of 0, 3, 10, or 20 mg/kg (30 mg/kg for 12 days, reduced to 20 mg/kg when dosing reinitiated on test day 22) for six months. In life measurements including hematology, clinical chemistry, tissue pathology (gross, microscopic, and organ weights), determination of selected hormone levels, cell proliferation, and bile-acid determinations were evaluated. To follow the movement of APFO/PFOA into and through the monkey, serum, urine, and fecal samples were collected at approximately two-week intervals and were analyzed for PFOA concentrations. Serum was collected from the femoral vein in the morning prior to daily dose administration. Urine and feces were obtained prior to morning dose administration following overnight collection in metabolism cages. At sacrifice (euthanasia with ketamine and xylazine), samples of liver tissue were weighed and flash frozen in liquid nitrogen and stored at minus 60 to 80°C until submitted for chemical analysis.

Intravenous Pharmacokinetic Study
Animals and husbandry. The three male and three female monkeys designated for use in this study were selected from an in-house colony of monkeys that were housed at Southern Research Institute (Southern Research) prior to use on this study. These monkeys were purchased from Charles River BRF, Inc. (Houston, TX) and were an estimated 3–4 years of age when placed on study.

Cage size and animal care conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals, 7th edition, and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99-198) and to the applicable Standard Operating Procedures (SOPs) of Southern Research. The Southern Research Institute Animal Care and Use Committee approved the study design. Southern Research Institute is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Certified, commercial, dry monkey chow #5048 (PMI Feeds, Inc., St. Louis, MO) was fed to the monkeys 2–3 times each day. The diet was supplemented with fresh fruit/treats several times each week. Tap water (Birmingham, Alabama public water supply) was available to the monkeys ad libitum. The monkeys were housed in a room that was maintained at a temperature of 20.0–21.3°C and a relative humidity of 22.2–65.7%. An automatic timer set to provide 12 h of light and 12 h of dark per day controlled room lights.

The monkeys used on the single iv exposure study had been used in prior single-dose iv pharmacokinetic studies of fluorochemicals. Specifically, they were previously given a single iv bolus doses of potassium perfluorobutanesulfonate (10 mg/kg); potassium perfluorobutanoate (10 mg/kg); and potassium perfluorohexanoate (10 mg/kg). PFOA was given after all three prior compounds were below the method quantitation limit (<0.004 µg/ml) which occurred within one month of dosing.

Materials and dose preparation. Potassium PFOA (linear, >99% pure by 19F NMR) was supplied by 3 M (St. Paul, MN). The test article was stored at room temperature until used. The vehicle used for the preparation of the dose formulation was sterile saline, USP (Phoenix Pharmaceutical Company; St. Joseph, MO; Lot 8070655). The vehicle was stored at room temperature. The formulation (5 mg/ml in saline) was stored refrigerated and used for dosing within four days after preparation; it was considered stable during this period. Dose concentration and homogeneity analyses were not performed.

Experimental design. On Day 0, each of the three male and three female monkeys received a single iv dose of potassium PFOA at 10 mg/kg into a superficial arm or leg vein. Doses were administered at a volume of 2 ml/kg. All monkeys were observed twice daily for clinical signs. Each monkey was weighed on Days –1, 4, 7, 14, 21, and 28. Urine was collected in standard metabolism cages for 24-h intervals on the following days: prior to dose administration (Day –3; baseline), on Day 1 (0–24 h postdose), on Day 2 (24–48 h postdose), and on Days 7, 14, 21, and 28. The volume of each urine sample was measured. Urine samples were stored frozen (approximately –20°C or below). Blood samples (approximately 3 ml) were collected from a superficial arm or leg vein of each monkey in a restraint chair at approximately 0 (predose) minutes; 0.5, 2, 4, 8, and 24 h; and on Days 2, 4, 7, 11, 14, 21, 28, 57, 79, 87, and 123 postdose. Samples were collected into tubes without anticoagulant and were allowed to clot at room temperature. The blood samples were then centrifuged, and the serum separated and stored frozen (approximately –20°C or below) until analyzed.

Analytical
Extraction of serum, urine, and liver samples. The analytical method for extraction and quantitative determination of PFOA from serum and liver samples has been published previously (Hansen et al., 2001Go). A study of PFOA binding to human plasma protein fractions was conducted, and PFOA was found to be <0.1% bound to fibrinogen (Kerstner-Wood et al., 2003Go); therefore, use of serum as a matrix should be representative of PFOA concentration in the non-cellular fraction of blood. Serum samples were extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into methyl-tert-butyl ether (MTBE). The MTBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 ml of methanol, then filtered through a 0.2 µm nylon filter using a 3 cc plastic syringe into glass autovials.

Liver samples were homogenized in water. An aliquot of each homogenate was spiked with surrogate and extracted and handled as described for the serum samples above.

For urine samples, the same ion-pairing LC/MS/MS methodology (as described by Hansen et al., 2001Go) was followed for the extraction and analysis of PFOA and is described briefly here. A small aliquot of the urine sample was mixed with a buffered solution containing tetrabutylammonium hydrogen sulfate, an ion-pairing reagent. After mixing, an organic solvent, either MTBE (six-month oral toxicity study) or ethyl acetate (iv study), based on laboratory validation, was added to the extract and shaken thoroughly. The sample was centrifuged prior to transferring the organic layer to a clean polypropylene tube and evaporated to dryness under a stream of nitrogen. The extract was reconstituted in methanol or in a methanol/water mixture and transferred to an autovial through a syringe filter.

Extraction of fecal samples. For fecal samples, approximately 1 g of sample was weighed into a polypropylene vial along with 10 ml of acetonitrile. The solution was capped and mixed on a wrist-action shaker for 30 min before being filtered through a glass acrodisk filter. The filtered extract was passed through a conditioned carbon solid-phase extraction (SPE) cartridge. The cartridge was eluted sequentially with 10 ml of acetonitrile followed by 20 ml of 90:10 acetonitrile:2% ascorbic acid in methanol. The combined extracts were evaporated to near dryness and reconstituted with methanol to a final volume of 2 ml.

Extraction efficiencies. It was not possible to verify true recovery of endogenous analyte from tissues because an isotopically labeled reference material was not available. The only measurement of accuracy available, matrix spike studies, indicate that the sera, liver, urine, and feces data can be considered to be accurate to within one SD of the average fortified sample recovery. The average fortified sample recovery was 93 ± 11%, 90 ± 26%, 88 ± 17%, and 117 ± 22%, respectively for sera, liver, urine, and feces.

Analysis of extracts. For all matrices, internal standard, either perfluorohexane sulfonate (C6F13SO3; PFHS) or tetrahydroperfluorooctanesulfonate (C10H4F17SO3; THPFOS), was added to each sample prior to the first extraction step. Method blanks, matrix blanks, and matrix spike samples were prepared along with each set of samples for quality control purposes. Standard curves used for quantitative determination of PFOA levels were spiked in and extracted from samples of blank primate matrix along with the samples. Each extraction process was validated for, at a minimum, accuracy, linearity, and method precision.

All extracts, regardless of matrix, were analyzed using liquid chromatography tandem mass spectrometry (LCMSMS), run in the negative-ion, electrospray mode. Separation was accomplished on a C18 LC column (Keystone Betasil/Aquasil or equivalent) using an ammonium acetate/methanol gradient. Quantitation was based on response of a fragment ion (either m/z = 169 or m/z = 369) collected after selection of the primary ion (m/z = 413) in the first quadrapole. Additional details can be found in Hansen et al. (2001)Go for on the LC/MS/MS conditions used.

Data quality. Based on the analysis of quality control samples and method validation, the accuracy of the urine data is estimated to be within the range of 60–140%; data collected on the remaining matrices is estimated to be within 70–130%. The lower limit of quantitation for PFOA determination in each matrix is: serum and urine = 0.010–0.020 µg/ml; liver and feces = 0.075 µg/g.

Data analyses. The serum concentration data for PFOA were subjected to non-compartmental pharmacokinetic analysis using WinNonlin (Standard Edition; Version 1.1; Scientific Consulting, Inc.; Cary, NC). Mean values and standard deviations for each parameter were calculated using Microsoft Excel software (Microsoft Corporation; Irvine, CA). The urinary excretion of PFOA at each collection interval was calculated and expressed as a percent of the administered dose. No other statistical analyses of the data were performed.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oral Pharmacokinetics from Six-Month Oral Toxicity Study
Serum concentrations. Individual serum concentrations during treatment are presented in Table 1 and all groups summarized in Figure 1. All treated groups reached apparent steady state within four weeks. A few of the early values in this time period appeared to be unusually high. After week 4, within dose groups intra- and interindividual values appeared to be reasonably consistent over time. The presence of PFOA in control monkeys ranged from not quantifiable to a maximum of approximately 0.4 µg/ml serum. There was a general reduction of values across all groups at week 18, which may represent an unexplained analytical phenomenon affecting all samples collected for Week 18. Steady-state concentrations of PFOA were 81 ± 40, 99 ± 50, and 156 ± 103 µg/ml serum for the 3, 10, and 20 mg/kg dose groups, respectively.


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TABLE 1 PFOA Concentration in Serum of Monkeys Receiving Daily Oral Doses of APFO

 


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FIG. 1. Mean PFOA concentration in serum of monkeys receiving daily oral dose of APFO. Means from weeks 2 and 4 of the 30/20 mg/kg dose group are not shown.

 
Mean serum PFOA values per group during treatment, taken as repeat measures, increased as dose increased but were not linearly proportional to dose (Fig. 2). Values in the high-dose group from six weeks on reflect the 20 mg/kg dose (not the initial 30 mg/kg dose) and are not believed to have been further influenced by initial dosing (see Butenhoff et al., 2002Go, for a discussion of effects from dosing with PFOs at 30 mg/kg).



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FIG. 2. Comparison of the average steady state concentrations in serum, urine, and feces of monkeys receiving daily oral dose of APFO.

 
Elimination of PFOA from serum after cessation of dosing in recovery monkeys from the 10 and 20 mg/kg dose groups is shown in Figure 3. For the two 10 mg/kg recovery monkeys, serum PFOA elimination half-life was 19.5 (R2 = 0.98) days and reflects first-order elimination kinetics. Similar half-lives were observed in the 20 mg/kg monkeys for whom dosing was suspended at various time points due to severe body-weight loss (see Butenhoff et al., 2002Go for a discussion of suspension of dosing for these monkeys). For these three 20 mg/kg monkeys, serum PFOA elimination half-life was 20.8 days (R2 = 0.82), again reflecting first-order elimination kinetics.



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FIG. 3. PFOA concentration in serum of recovery monkeys receiving daily oral dose of APFO. Open symbols 0 mg/kg; closed symbols 10 mg/kg; crosses 20 mg/kg.

 
Urine PFOA concentrations. Table 2 presents mean urine PFOA concentrations per dose group during treatment. As with serum PFOA concentrations, there appeared to be relatively high values in the week 2 and week 4 samples; however, urine PFOA concentrations after week 4 appeared to be at steady state. Urine PFOA concentrations were noticeably less in the 3 mg/kg dose group compared to the 10 and 20 mg/kg dose groups. Overall mean concentrations for the 3, 10, and 20 mg/kg day dose groups were 53 ± 25, 166 ± 83, and 181 ± 100 µg/ml, respectively. Many of the individual urine PFOA concentrations for the 3 mg/kg dose group were less than the quantitation limit (0.02 µg/ml). Although urine PFOA concentrations in the 10 mg/kg dose group were less on average than those in the 20 mg/kg dose group, this difference was small and was not proportional to the difference in administered dose.


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TABLE 2 PFOA Concentration in Urine of Monkeys Receiving Daily Oral Doses of APFO

 
Figure 4 shows the reduction in urine PFOA concentrations over time in recovery monkeys. In the 10 and 20 mg/kg recovery monkeys, within two weeks of recovery, urine PFOA concentrations were <1% of the previous value measured during treatment, after which they decreased relatively slowly.



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FIG. 4. Mean PFOA concentration in urine of recovery monkeys receiving daily oral dose of APFO. Open symbols 0 mg/kg; closed symbols 10 mg/kg; crosses 20 mg/kg.

 
Fecal PFOA concentrations. Mean feces concentrations of PFOA during treatment are shown in Table 3. From week 2 forward, the fecal PFOA concentrations are relatively consistent within treatment groups and increase in approximate linear proportionality between dose groups. Overall mean concentrations in the 3, 10, and 20 mg/kg dose groups were 6.8 ± 5.3, 28 ± 20, and 50 ± 33, respectively. Within two weeks of recovery, fecal APFO concentrations dropped to less than 10% of the last measured value during treatment, and thereafter declined slowly (Fig. 5).


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TABLE 3 PFOA Concentration in Feces of Monkeys Receiving Daily Oral Doses of APFO

 


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FIG. 5. Mean PFOA concentration in feces of recovery monkeys receiving daily oral dose of APFO. Open symbols 0 mg/kg; closed symbols 10 mg/kg; crosses 20 mg/kg.

 
Liver PFOA concentrations. Liver PFOA concentrations measured in the monkeys are represented in Table 4. As with serum, the PFOA concentrations in liver did not increase in linear proportion to dose. The values at terminal sacrifice just after dosing for the 3 mg/kg and 10 mg/kg dose groups were similar, and ranged from 6.29 to 21.9 µg PFOA/g tissue. The 20 mg/kg day dose-group values for the two monkeys sacrificed on week 27 were 16.0 and 83.3 µg PFOA/g tissue. It is noteworthy that the highest liver value obtained (154 µg PFOA/g tissue) was from the monkey sacrificed in extremis on week 5. Liver from monkeys at the end of the recovery period contained 0.08 and 0.15 µg PFOA/g, approaching levels seen in the controls.


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TABLE 4 PFOA Concentration in Livers of Monkeys Receiving Daily Doses Orally of APFO

 
Intravenous Pharmacokinetic Study
Mortality, clinical observations, and body weights. One of three male monkeys (2052) was euthanized on Day 79 because of repeated episodes of self-mutilation; there was no indication that the self-mutilation was related to administration of potassium PFOA and no adverse clinical signs of toxicity were observed for any monkey. This male monkey 2052 was observed on Day 57 of the study to have several wounds on the right leg. The attending veterinarian diagnosed these wounds to be self-inflicted lacerations. The wounds were cleaned and subsequently flushed with a weak betadine solution every 3–4 days for four courses of treatment. The social enrichment of the monkey was also augmented in order to provide the monkey additional stimulation and to change his focus of attention. On Day 79, the monkey was examined and several severe lacerations were observed on the leg and foot of the animal. These lacerations were again diagnosed as self-inflicted wounds. Due to the repeated instances of self-mutilation and the likelihood of continued episodes of self-mutilation, the monkey was euthanized on Day 79 for humane reasons. After completion of the study, serum cortisol, as a potential marker of stress, was measured in reserve samples from all monkeys, and there was no indication that serum cortisol for male monkey 2052 was different from his male and female peers (data not shown).

No test article-related deaths occurred during the study. The body weight of each monkey remained essentially the same between pre-test day 1 through test day 28 (data not shown).

Serum and urine analysis. Serum concentrations of PFOA in three male and three female monkeys at various times through Day 123 after administration of a single iv dose of 10 mg/kg are presented in Table 5. At 0.5 h after dosing, serum concentrations of PFOA ranged from 91 to 97 µg/ml in the male monkeys and from 89 to 96 µg/ml in the female monkeys. At 48 h, serum concentrations of PFOA were between 49 and 66 µg/ml in the male monkeys and 40 and 62 µg/ml in the female monkeys. Beyond this time, serum concentrations of PFOA in two of the three male monkeys decreased at a somewhat faster rate than that observed for the third male monkey and for the three female monkeys. On Day 28, the concentration of PFOA in serum ranged from 1.9 to 27 µg/ml in the male monkeys and from 7.1 to 34 µg/ml in the female monkeys. Due to these relatively high serum concentrations of PFOA on this day, the length of serum collection was extended through Day 123. On Day 123, the serum concentration of PFOA was at or only slightly above the limit of quantitation (0.02 µg/ml) in the two surviving male monkeys and between 0.89 and 4.7 µg/ml in the three female monkeys.


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TABLE 5 PFOA Concentration in Serum of Monkeys Receiving a Single iv Dose of Potassium PFOA

 
The amount of PFOA eliminated in urine by individual monkeys at various times after dosing is presented in Table 6. Sex differences in the urinary excretion of PFOA were not readily apparent prior to Day 21. On Days 21 and 28, the female monkeys appeared to eliminate a somewhat higher percentage of the dose in urine than did the male monkeys. The urinary elimination of PFOA was prolonged for both the male and female monkeys; for example, on Day 28 from 0.1 to 1% of the administered dose was recovered in urine collected from the individual monkeys.


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TABLE 6 PFOA Concentration in Urine of Monkeys Receiving a Single iv Dose of APFO

 
Pharmacokinetic parameters. Pharmacokinetic parameters calculated from serum concentrations of PFOA in individual monkeys from the iv study and by group for the six-month oral study are presented in Table 7. For the iv study, the values were derived from non-compartmental pharmacokinetic analysis of the data. AUC0–infinity values ranged from 571 to 2501 µg·day/ml (mean: 1235 µg·day/ml) for male monkeys and from 1094 to 3224 µg·day/ml (mean 2293 µg·day/ml) for female monkeys. The terminal half-lives of PFOA in serum were 13.6, 13.7, and 35.3 days in the three male monkeys and 26.8, 29.3, and 41.7 days in the three female monkeys. The total body clearences were 4.0, 15.8, and 17.5 ml/day/kg for male monkeys and 3.1, 3.9, and 9.1 ml/day/kg for female monkeys. These estimated values for half-life and clearance indicated that two of the three male monkeys eliminated PFOA at a faster rate than did the female monkeys. The volumes of distribution of PFOA at steady state (Vdss) were similar for both sexes and ranged from 168, 184, to 192 ml/kg for the male monkeys and 133, 190, to 270 ml/kg for the female monkeys.


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TABLE 7 Pharmacokinetic Parameters Calculated from Serum Concentrations of PFOA of Monkeys Receiving a Single iv Dose of APFO or Repeated Oral Doses of PFOA

 
Mean serum elimination half-lives for the 10 mg/kg and 20 mg/kg dose group recovery males in the six-month oral study were 19.5 and 20.8 days, respectively (Table 7). Recovery data was not obtained for the 3 mg/kg dose-group males.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Results of both the oral dosing and intravenous dosing studies provided sufficient information to characterize the movement of PFOA through monkeys following either repeated oral dosing or single iv dosing. From the data obtained in the iv study, it was possible to establish classic pharmacokinetic parameters, including Cmax, Cpss, Vdss, Kd, AUC, and clearance. Although the oral study was not designed to obtain a mass balance or establish pharmacokinetic parameters, sampling of excretory products (every two weeks) allowed estimation of absorption, steady-state values, and excretion patterns as a function of both dose and time. PFOA was occasionally found in untreated controls at amounts that were near or not largely above the LOQ (limit of quantitation). The finding of PFOA in controls suggests cross-contamination and/or environmental exposure (food/water/air) but does not affect the ability to draw conclusions from the data. In the oral study, treatment groups were housed separately from the control group. In the iv study, monkeys were kept in separate cages in the same room.

Oral Study
The oral study design included provision for two male monkeys to enter a recovery period after six months of treatment in the 0, 10, and 30/20 mg/kg. Due to poor tolerance and one unscheduled sacrifice, treatment needed to be discontinued in four monkeys in the 30/20 mg/kg dose group prior to the scheduled six-month dosing period. Thus, three monkeys in this group became de facto recovery monkeys and were sampled at two-week intervals. As a result, only two monkeys in this dose group were followed for the entire six months of APFO treatment. The only instance where a sample size of two is felt to have hampered data interpretation was in the liver tissue concentration in which the two monkeys dosed through six months showed widely different liver tissue values (16 and 83 µg/g).

Intra- and interindividual variation was expected and was seen in these monkeys. However, the magnitude of this variability is not unexpectedly large and appears reasonably consistent over time. To an extent, this variability is likely a result of analytical variability (up to ±30%). Considering that within group values are within a consistent range over time, it was appropriate to treat these values as repeat measures to obtain overall group mean values (Table 1). The greatest variability was seen in the 30/20 mg/kg dose group.

Serum samples from monkeys receiving APFO orally reached apparent steady-state levels very quickly, within four weeks of treatment (Table 1). PFOA blood concentration increased with dose but not in a linear manner. The mean serum values at steady state for the 3, 10, and 20 mg/kg dose groups form an approximate 1:1:2 ratio where the dose ratio is approximately 1:3:7. The analysis of these data is affected by the fact that the total number of monkeys carried through six months treatment at 20 mg/kg was only two.

Urinary PFOA concentrations also appeared to reach steady-state very quickly with values from 4 weeks through 26 weeks appearing similar in each of the three treatment groups. A ratio of approximately 1:3:4 was seen in mean urine concentrations as the administered dose ratio was approximately 1:3:7. Again, the number of monkeys on continuous treatment for six months at 20 mg/kg was only two, which affects analysis of the data.

Fecal PFOA concentrations were similar from treatment week 4 through the end of the six-month dosing period in all three treatment groups. A ratio of approximately 1:4:7 was seen in the groups given oral doses in the ratio of approximately 1:3:7. Thus, the fecal concentration appears to follow the delivered dose ratio more closely than do either the serum or urinary PFOA concentrations.

Although urinary volume and fecal mass were not measured, using estimated daily fecal and urine quantities, a calculated 6.8, 21.7, and 23.8 mg were excreted daily in the urine, and 0.13, 0.53, and 0.95 mg in the feces. The excreted estimates came from data describing a standard cynomolgus male monkey (Suzuki et al., 1989Go). If the estimated mg amounts excreted are calculated in terms of mg/kg, the excreted PFOA tends to be considerably more than the amount of APFO given orally per day (7 ± 4, 22 ± 11, and 30 ± 26 mg/kg, respectively for the 3, 10, and 20 mg/kg dose groups). Again, obtaining a material balance was not a goal of this study; however, it is apparent that urinary excretion is the main route of elimination of PFOA in the cynomolgus monkey.

Serum levels of PFOA dropped significantly upon cessation of dosing in the monkeys either designated for recovery or removed from treatment due to poor tolerance of the chemical. Although the elimination appears to be first order, the elimination profile of the two 10 mg/kg monkeys during recovery could be interpreted as biphasic with an elimination half-life of approximately one week during weeks 26 to 30 and approximately three weeks during weeks 30 to 40. Similar declines were seen in the 20 mg/kg monkeys removed from treatment. Urinary PFOA concentrations also dropped dramatically from 109 µg/ml at 26 weeks to 0.32 µg/ml two weeks into recovery to 0.03 µg/ml at 12 weeks recovery. Fecal PFOA concentrations also dropped dramatically upon cessation of APFO dosing with mean concentrations of 43 µg/ml in 10 mg/kg monkeys on week 26 falling to 0.38 µg/ml 2 weeks later, to 0.034 ppm from 10 to 12 weeks later. Thus, PFOA appears to clear in male cynomolgus monkeys upon cessation of daily oral dosing with PFOA with a serum elimination half-life of approximately 20 days.

Intravenous Pharmacokinetic Study
PFOA was eliminated by male and female monkeys given a single iv dose of 10 mg/kg. The results of this study indicated that beginning on around Day 7 and continuing throughout the remaining period of sample collection, serum concentrations of PFOA were lower in two of the three male monkeys than in the three female monkeys. Therefore, the clearance of PFOA appeared to be somewhat higher in two of the male monkeys than in the three female monkeys. Although the number of monkeys was limited, these results suggest that the elimination kinetics of PFOA may be slightly different in male and female cynomolgus monkeys. However, given the small number of animals, it is not possible to conclude a significant sex difference with regard to PFOA elimination. It is worthy to note that the range of serum PFOA elimination half-lives (males and females) was 14 to 42 days, which includes the calculated serum elimination half-life values of approximately 20 days seen in the male monkeys in the six-month oral toxicity study.

Although the serum levels of PFOA indicated that at least two of the three male monkeys may have eliminated PFOA at a faster rate than did the female monkeys, it was difficult to discern a difference in the urinary excretion of the compound by male and female monkeys. This may have been related to the fact that the rate of urinary excretion of PFOA by all the monkeys on study was slow. Less than 20% of the administered dose was excreted in urine by either male or female monkeys within the first 48 h after dosing.

The serum PFOA elimination half-life for the male monkey (number 2052) that was euthanized because of self-mutilation more closely paralleled the serum PFOA elimination half-lives observed for the female monkeys than that observed for the other two male monkeys. The possibility was considered that the self-mutilation activities of this monkey, which led to the eventual euthanasia for humane reasons, was a cause of or a response to stress that could have affected elimination of PFOA. Post-study analysis of serum cortisol in retained serum samples from all monkeys did not indicate noticeable differences between this monkey and his study peers. It is possible that the variation seen was a reflection of individual variation in PFOA elimination kinetics.

The results for the male cynomolgus monkeys from the iv pharmacokinetics study would predict that steady state in the daily oral dosing study would not be reached until at least eight weeks of daily oral dosing. The fact that serum PFOA concentrations did not increase after four weeks of daily oral dosing at all treatment levels suggests either a shorter elimination half-life than would be anticipated by the iv study or other factors that may reduce anticipated steady-state serum PFOA concentrations. Because the elimination of PFOA from male monkeys after cessation of treatment in the daily oral dosing study was observed to have a half-life of approximately 20 days as compared to 14 to 36 days in the iv study, it is not likely that elimination is the limiting factor in reducing predicted steady state serum PFOA concentrations. Possible explanations for this discrepancy include incomplete oral absorption and entrapment of a portion of oral dose in enterohepatic circulation. Oral absorption has been shown to be essentially complete in rats (Gibson and Johnson, 1979Go; Kennedy et al., 2004Go), and PFOA is known to enter enterohepatic circulation in the rat (Johnson et al., 1984Go). Steady-state fecal concentrations were approximately linear in proportion to dose during treatment in the oral study, in contrast to serum and urine concentrations, which did not increase in linear proportion to dose. After cessation of treatment, fecal concentrations fell sharply within two weeks to 10% of steady state concentrations, after which they decreased more slowly. This observation suggests both incomplete absorption at each dose level and fecal elimination of a portion of PFOA recycled in bile. This may explain lower steady-state serum PFOA concentrations than would have been predicted from the iv data.

The volume of distribution at steady state determined in the iv study (133–270 ml/kg) suggests distribution primarily in extracellular space. Similar Vdss values (values, respectively) have been determined in male and female rats (Kemper, 2003Go), and several studies on the tissue distribution of PFOA in rats suggest that most of the delivered dose is in blood, liver, and kidney (Kemper, 2003Go; Vanden Heuvel et al., 1991Go). The fact that PFOA is not known to be metabolized (Johnson et al., 1984Go; Vanden Heuvel et al., 1991Go) suggests that there should not be large differences between species. Steady state serum concentrations in male rats given oral doses in the same range as the monkeys in the six-month oral study are similar in magnitude (Butenhoff et al., 2004aGo,bGo; Kennedy et al., 2004Go).

In conclusion, during repeated oral dosing, PFOA reaches a steady concentration in the serum, urine, and feces within four weeks with concentrations increasing with dose in a nonlinear manner. A sex dependence of PFOA pharmacokinetics in monkeys was not clear from the iv study. Based on the results of both the oral and iv studies, urine was the primary route of excretion with an elimination half life of approximately 20–30 days. The PFOA elimination half life following either oral or intravenous dosing is approximately 20–30 days.


    NOTES
 

1 To whom correspondence should be addressed at 3M Center 220-2E-02, St. Paul, MN 55144. Fax: (651) 733-1733. E-mail: jlbutenhoff{at}mmm.com.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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