Mark J. Strynar

Modeling Single and Repeated Dose Pharmacokinetics of PFOA in Mice

Perfluorooctanoic acid (PFOA) displays complicated pharmacokinetics in that serum concentrations indicate long half-lives despite which steady state appears to be achieved rapidly. In this study, serum and tissue concentration time-courses were obtained for male and female CD1 mice after single, oral doses of 1 and 10 mg/kg of PFOA. When using one- and two-compartment models, the pharmacokinetics for these two dosages are not consistent with serum time-course data from female CD1 mice administered 60 mg/kg, or with serum concentrations following repeated daily doses of 20 mg/kg PFOA. Some consistency between dose regimens could be achieved using the saturable resorption model of Andersen et al. In this model PFOA is cleared from the serum into a filtrate compartment from which it is either excreted or resorbed into the serum by a process presumed transporter mediated with a Michaelis-Menten form. Maximum likelihood estimation found a transport maximum of T(m) = 860.9 (1298.3) mg/l/h and half-maximum concentration of K(T) = 0.0015 (0.0022) mg/l where the estimated standard errors (in parentheses) indicated large uncertainty. The estimated rate of flow into and out of the filtrate compartment, 0.6830 (1.0131) l/h was too large to be consistent with a biological interpretation. For these model parameters a single dose greater than 40 mg/kg, or a daily dose in excess of 5 mg/kg were necessary to observe nonlinear pharmacokinetics for PFOA in female CD1 mice. These data and modeling analyses more fully characterize PFOA in mice for purposes of estimating internal exposure for use in risk assessment.

Prenatal Perfluorooctanoic Acid Exposure in CD-1 Mice: Low-Dose Developmental Effects and Internal Dosimetry

Perfluorooctanoic acid (PFOA) is an environmental contaminant that causes adverse developmental effects in laboratory animals. To investigate the low-dose effects of PFOA on offspring, timed-pregnant CD-1 mice were gavage dosed with PFOA for all or half of gestation. In the full-gestation study, mice were administered 0, 0.3, 1.0, and 3.0 mg PFOA/kg body weight (BW)/day from gestation days (GD) 1-17. In the late-gestation study, mice were administered 0, 0.01, 0.1, and 1.0 mg PFOA/kg BW/day from GD 10-17. Exposure to PFOA significantly (p < 0.05) increased offspring relative liver weights in all treatment groups in the full-gestation study and in the 1.0 mg PFOA/kg group in the late-gestation study. In both studies, the offspring of all PFOA-treated dams exhibited significantly stunted mammary epithelial growth as assessed by developmental scoring. At postnatal day 21, mammary glands from the 1.0 mg/kg GD 10-17 group had significantly less longitudinal epithelial growth and fewer terminal end buds compared with controls (p < 0.05). Evaluation of internal dosimetry in offspring revealed that PFOA concentrations remained elevated in liver and serum for up to 6 weeks and that brain concentrations were low and undetectable after 4 weeks. These data indicate that PFOA-induced effects on mammary tissue (1) occur at lower doses than effects on liver weight in CD-1 mice, an observation that may be strain specific, and (2) persist until 12 weeks of age following full-gestational exposure. Due to the low-dose sensitivity of mammary glands to PFOA in CD-1 mice, a no observable adverse effect level for mammary developmental delays was not identified in these studies.

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.

Previous studies in mice with multiple gestational exposures to perfluorooctanoic 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 5mg 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 postnatal day 8, regardless of dose. The 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.

Relative importance of wastewater treatment plants and non-point sources of perfluorinated compounds to Washington State rivers

Perfluorinated compounds (PFCs) were measured in 10 Washington State rivers and 4 wastewater treatment plants (WWTPs) under periods of low and high flows to investigate the relative importance of point and non-point sources to rivers. PFCs were detected in all samples with summed values ranging from 1.11 to 74.9 ng/L in surface waters and 62.3-418 ng/L in WWTP effluent. Concentrations in 6 of the 10 rivers exhibited a positive relationship with flow, indicating runoff as a contributing source, with PFC loads greatest at all 10 waterbodies during high flows. Perfluoroheptanoic acid:perfluorooctanoic acid homologue ratios suggest atmospheric contributions to the waterbodies are important throughout the year. Principal component analysis (PCA) indicated distinct homologue profiles for high flow, low flow, and effluent samples. The PCA demonstrates that during the spring when flows and loads are at their greatest; WWTP discharges are not the primary sources of PFCs to the river systems. Taken together, the evidence provided signifies non-point inputs are a major pathway for PFCs to surface waters in Washington State.

Dosimetric Anchoring of In Vivo and In Vitro Studies for Perfluorooctanoate and Perfluorooctanesulfonate

In order to compare between in vivo toxicity studies, dosimetry is needed to translate study-specific dose regimens into dose metrics such as tissue concentration. These tissue concentrations may then be compared with in vitro bioactivity assays to perhaps identify mechanisms relevant to the lowest observed effect level (LOEL) dose group and the onset of the observed in vivo toxicity. Here, we examine the perfluorinated compounds (PFCs) perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS). We analyzed 9 in vivo toxicity studies for PFOA and 13 in vivo toxicity studies for PFOS. Both PFCs caused multiple effects in various test species, strains, and genders. We used a Bayesian pharmacokinetic (PK) modeling framework to incorporate data from 6 PFOA PK studies and 2 PFOS PK studies (conducted in 3 species) to predict dose metrics for the in vivo LOELs and no observed effect levels (NOELs). We estimated PK parameters for 11 combinations of chemical, species, strain, and gender. Despite divergent study designs and species-specific PK, for a given effect, we found that the predicted dose metrics corresponding to the LOELs (and NOELs where available) occur at similar concentrations. In vitro assay results for PFOA and PFOS from EPAs ToxCast project were then examined. We found that most in vitro bioactivity occurs at concentrations lower than the predicted concentrations for the in vivo LOELs and higher than the predicted concentrations for the in vivo NOELs (where available), for a variety of nonimmunological effects. These results indicate that given sufficient PK data, the in vivo LOELs dose regimens, but not necessarily the effects, could have been predicted from in vitro studies for these 2 PFCs.

Developmental toxicity of perfluorononanoic acid in mice

Perfluorononanoic acid (PFNA) is a ubiquitous and persistent environmental contaminant. Although its levels in the environment and in humans are lower than those of perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA), a steady trend of increases in the general population in recent years has drawn considerable interest and concern. Previous studies with PFOS and PFOA have indicated developmental toxicity in laboratory rodent models. The current study extends the evaluation of these adverse outcomes to PFNA in mice. PFNA was given to timed-pregnant CD-1 mice by oral gavage daily on gestational day 1-17 at 1, 3, 5 or 10mg/kg; controls received water vehicle. Dams given 10mg/kg PFNA could not carry their pregnancy successfully and effects of this dose group were not followed. Similar to PFOS and PFOA, PFNA at 5mg/kg or lower doses produced hepatomegaly in the pregnant dams, but did not affect the number of implantations, fetal viability, or fetal weight. Mouse pups were born alive and postnatal survival in the 1 and 3mg/kg PFNA groups was not different from that in controls. In contrast, although most of the pups were also born alive in the 5mg/kg PFNA group, 80% of these neonates died in the first 10 days of life. The pattern of PFNA-induced neonatal death differed somewhat from those elicited by PFOS or PFOA. A majority of the PFNA-exposed pups survived a few days longer after birth than those exposed to PFOS or PFOA, which typically died within the first 2 days of postnatal life. Surviving neonates exposed to PFNA exhibited dose-dependent delays in eye opening and onset of puberty. In addition, increased liver weight seen in PFNA-exposed offspring persisted into adulthood and was likely related to the persistence of the chemical in the tissue. Evaluation of gene expression in fetal and neonatal livers revealed robust activation of peroxisome proliferator-activated receptor-alpha (PPARα) target genes by PFNA that resembled the responses of PFOA. Our results indicate that developmental toxicity of PFNA in mice is comparable to that of PFOS and PFOA, and that these adverse effects are likely common to perfluoroalkyl acids that persist in the body.

Estimating common parameters of lognormally distributed environmental and biomonitoring data: Harmonizing disparate statistics from publications

The progression of science is driven by the accumulation of knowledge and builds upon published work of others. Another important feature is to place current results into the context of previous observations. The published literature, however, often does not provide sufficient direct information for the reader to interpret the results beyond the scope of that particular article. Authors tend to provide only summary statistics in various forms, such as means and standard deviations, median and range, quartiles, 95% confidence intervals, and so on, rather than providing measurement data. Second, essentially all environmental and biomonitoring measurements have an underlying lognormal distribution, so certain published statistical characterizations may be inappropriate for comparisons. The aim of this study was to review and develop direct conversions of different descriptions of data into a standard format comprised of the geometric mean (GM) and the geometric standard deviation (GSD) and then demonstrate how, under the assumption of lognormal distribution, these parameters are used to answer questions of confidence intervals, exceedance levels, and statistical differences among distributions. A wide variety of real-world measurement data sets was reviewed, and it was demonstrated that these data sets are indeed of lognormal character, thus making them amenable to these methods. Potential errors incurred from making retrospective estimates from disparate summary statistics are described. In addition to providing tools to interpret “other people’s data,” this review should also be seen as a cautionary tale for publishing one’s own data to make it as useful as possible for other researchers.

Partial life-cycle and acute toxicity of perfluoroalkyl acids to freshwater mussels

Freshwater mussels are among the most sensitive aquatic organisms to many contaminants and have complex life-cycles that include several distinct life stages with unique contaminant exposure pathways. Standard acute (24-96 h) and chronic (28 d) toxicity tests with free larva (glochidia) and juvenile mussels are effective at generating data on contaminant effects at two discrete life stages but do not incorporate effects on brooded glochidia. We developed a novel partial life-cycle assay that incorporates exposures to brooding adult female mussels and used this method in combination with acute toxicity tests to assess adverse effects of perfluoroctanesulfonic acid (PFOS) and perfluoroctanoic acid (PFOA) on freshwater mussels. Fatmucket (Lampsilis siliquoidea) were exposed to PFOS at two life stages: brooding glochidia (in marsupia) for 36 d and free glochidia in water for 24 h. In standard acute tests with glochidia (24-48 h exposures) and juveniles (48-96 h exposures) of fatmucket and black sandshell (Ligumia recta), glochidia were 8 to 25 times more sensitive than juveniles. Perfluoroctanesulfonic acid significantly reduced the duration of glochidia viability and reduced probability of metamorphosis at concentrations 3,000 times lower than the most sensitive acute endpoint (24-h EC50). The partial life-cycle test is adaptable to a variety of endpoints and research objectives and is useful for identifying adverse effects at contaminant concentrations below those required for an acute lethal response.

Evaluating an Alternative Method for Rapid Urinary Creatinine Determination

Creatinine (CR) is an endogenously produced chemical that is routinely assayed in urine specimens to assess kidney function and sample dilution. The industry-standard method for CR determination, known as the kinetic Jaffé (KJ) method, relies on an exponential rate of a colorimetric change, and can therefore require automated processing equipment for moderate- to high-throughput analysis (hundreds to thousands of samples per day). This study evaluates an alternative colorimetric method, the “plateau Jaffé” (PJ) method, which utilizes the chemistry of the KJ method, a commercially available kit, and a multipoint calibration curve. This method is amenable to moderate-throughput sample analysis and does not require automated processing equipment. Thirty-two spot urine samples from healthy adult volunteers were analyzed for creatinine concentration (CRc) using the KJ and PJ methods. Samples were also analyzed using a liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) method, which acted as an analytical control. Replicate measurements of spot samples (natural log-transformed values) were used to estimate method precision, and linear regression models were used to evaluate method accuracy (LC-TOF/MS measurements were considered the analytical benchmark). Measurement precision was comparable across all three methods, with coefficent of variation estimates ranging from 3 to 6%. Regression models generally showed good agreement across methods with R2 estimates ranging from .996 to .998, slope estimates ranging from .944 to .986, and y-intercept estimates ranging from 0.111 to 0.303. Minor bias (between 2 and 16%) was observed across methods at the tails of the measurement distributions. The provided regression equations can be used to adjust for this bias and to improve CR measurement comparisons across studies employing different methods. Considering these results, the PJ method is a suitable alternative to the industry standard KJ method for urinary CRc determination. It can be implemented for moderate-throughput sample analysis using modest and commonly available lab instrumentation and manual sample preparation techniques.

Evaluation of the Immunomodulatory Effects of 2,3,3,3-Tetrafluoro-2-(Heptafluoropropoxy)-Propanoate in C57BL/6 Mice

2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate was designed to replace perfluorooctanoic acid (PFOA), which has been mostly phased out of U.S. production due to environmental persistence, detectable human and wildlife serum concentrations, and reports of systemic toxicity. In rodent models, PFOA exposure suppresses T cell-dependent antibody responses (TDAR) and vaccine responses in exposed humans. To determine replacement compound effects on TDAR and related parameters, male and female C57BL/6 mice were gavaged with 0, 1, 10, or 100u2009mg/kg/day for 28 days. Mice immunized with antigen on day 24 were evaluated for TDAR and splenic lymphocyte subpopulations five days later. Serum and urine were collected for test compound concentrations and liver peroxisome proliferation was measured. Relative liver weight at 10 and 100u2009mg/kg and peroxisome proliferation at 100u2009mg/kg were increased in both sexes. TDAR was suppressed in females at 100u2009mg/kg. T lymphocyte numbers were increased in males at 100u2009mg/kg; B lymphocyte numbers were unchanged in both sexes. Females had less serum accumulation and higher clearance than males, and males had higher urine concentrations than females at all times and doses. While this PFOA-replacement compound appears less potent at suppressing TDAR relative to PFOA, it produces detectable changes in parameters affected by PFOA; further studies are necessary to determine its full immunomodulatory profile and potential synergism with other per- and polyfluoroalkyl substances of environmental concern.2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate was designed to replace perfluorooctanoic acid (PFOA), which has been mostly phased out of U.S. production due to environmental persistence, detectable human and wildlife serum concentrations, and reports of systemic toxicity. In rodent models, PFOA exposure suppresses T cell-dependent antibody responses (TDAR) and vaccine responses in exposed humans. To determine replacement compound effects on TDAR and related parameters, male and female C57BL/6 mice were gavaged with 0, 1, 10, or 100u2009mg/kg/day for 28 days. Mice immunized with antigen on day 24 were evaluated for TDAR and splenic lymphocyte subpopulations five days later. Serum and urine were collected for test compound concentrations and liver peroxisome proliferation was measured. Relative liver weight at 10 and 100u2009mg/kg and peroxisome proliferation at 100u2009mg/kg were increased in both sexes. TDAR was suppressed in females at 100u2009mg/kg. T lymphocyte numbers were increased in males at 100u2009mg/kg; B lymphocyte numbers were unchanged in both sexes. Females had less serum accumulation and higher clearance than males, and males had higher urine concentrations than females at all times and doses. While this PFOA-replacement compound appears less potent at suppressing TDAR relative to PFOA, it produces detectable changes in parameters affected by PFOA; further studies are necessary to determine its full immunomodulatory profile and potential synergism with other per- and polyfluoroalkyl substances of environmental concern.