* Biomathematics Graduate Program, Department of Statistics, North Carolina State University, Raleigh, North Carolina 27695; and
Chemical Industry Institute of Toxicology Research Triangle Park, North Carolina 27709
Received February 2, 1999; accepted August 26, 1999
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ABSTRACT |
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Key Words: phthalates; DBP; MBP; PBPK; model; flow-limited; diffusion-limited; pH trapping; enterohepatic circulation; log-likelihood ratio test..
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INTRODUCTION |
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Several phthalate esters, including DBP, are testicular toxicants in rodents at high doses. Oral exposure of adult or juvenile male rats for 4 to 90 days to 5002600 mg/kg/day DBP resulted in decreased testicular weight, atrophy of the seminiferous tubules, and decreased sperm counts (Cater et al., 1977; Fukuoka et al., 1993
; Fukuoka et al., 1989
; Fukuoka et al., 1990
; Gray et al., 1982
; Srivastava et al., 1990
). These testicular effects have been shown to be partly reversible (Tanino et al., 1987
) and are species sensitive, with rat and guinea pigs more sensitive than mice and hamsters (Gray et al., 1982
; NTP, 1995
; Oishi and Hiraga, 1980
). Pubertal rats are also more sensitive than adult rats to the toxicity of phthalate esters (Sjöberg et al., 1986
). Dietary administration of doses equivalent to 66 mg/kg/day throughout gestation to adulthood resulted in malformation of the male reproductive tract (NTP, 1991
; Wine et al., 1997
), as did gestational and lactational exposure alone to oral doses of 250 mg/kg/day DBP and higher (Mylchreest et al., 1998
). The presence of malformations at lower doses in perinatal rats than in adult males indicates that the period of time encompassing gestation and lactation is the most sensitive exposure window for developmental effects due to phthalate ester exposure.
Orally administered phthalate diesters are rapidly metabolized in the gut to monoesters by pancreatic lipase and gut flora. DBP is metabolized to mono(n-butyl) phthalate (MBP). At doses up to 857 mg/kg DBP, no intact DBP was measured in the blood of exposed rats (NIEHS, 1994), indicating that virtually no intact DBP was absorbed. MBP is further metabolized by
and
- 1 oxidation in the rat liver (Albro and Moore, 1974
; Williams and Blanchfield, 1975
). MBP also undergoes glucoronide conjugation in the rat, and both free and conjugate MBP are excreted in the urine (Foster et al., 1983
). There is no evidence of tissue accumulation of DBP or its metabolites. Radioactivity was detected in liver, kidney, blood, muscle, adipose tissue, and the intestine 24 h after administration of a single oral dose of 60 mg/kg DBP; however, 90% of the administered radioactivity was excreted in the urine by 48 h (Tanaka et al., 1978
).
The phthalate monoester appears to be the active toxicologic compound for the induction of reproductive toxicity (Gray and Gangolli, 1986; Oishi and Hiraga, 1980
). MBP induces testicular atrophy after similar oral doses
400 mg/kg MBP as DBP with the same spectrum of effects (Cater et al., 1977
; Foster et al., 1981
; Gray et al., 1982
; Oishi and Hiraga, 1980
).
A physiologically based pharmacokinetic (PBPK) model was developed previously for di(2-ethylhexyl) phthalate (DEHP) and its monoester, MEHP (Keys et al., 1999). In that study, several relatively complicated PBPK model structures (enterohepatic circulation, diffusion-limitation, and pH trapping) were compared to a simpler flow-limited model. The pH trapping model provided the best description of DEHP and MEHP dosimetry. The objective of the present study was to develop a PBPK model for DBP/MBP and to simultaneously test the applicability of the DEHP and MEHP PBPK model structure to the less lipophilic phthalate diester DBP and its monoester MBP. The parameterization of the DEHP/MEHP model for DBP/MBP included laboratory measurement of MBP tissue:blood partition coefficients. A combined diffusion-limited and pH trapping model that had not been examined in the context of previous work on DEHP/MEHP was also examined in the present study.
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METHODS |
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Animals.
This study was conducted under federal guidelines for the care and use of laboratory animals (NRC, 1996) and was approved by the CIIT Institutional Animal Care and Use Committee. Ten-week-old male CD rats (Sprague-Dawley) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Animals were housed individually in solid-bottom plastic cages with Alpha Dri bedding under standard conditions of temperature, humidity, and light. Rodent feed (NIH-07) and deionized water via water bottles were provided ad libitum. The rats were acclimated 6 days or more prior to use.
Rats were euthanized with CO2, and whole blood was collected from the vena cava using a heparinized syringe. Liver, muscle, fat, and testes were then removed and stored at -80° until needed for measurement of tissue:blood partition coefficients.
Measured partition coefficients.
Tissue:propylene carbonate (PC) partition coefficients were determined for MBP using a vial equilibration method developed for nonvolatile chemicals (Murphy et al., 1995). Radiolabeled MBP was equilibrated between tissue homogenates, saline or whole blood and saline-saturated PC at 37°C. Aliquots of PC were taken at 2 time points to ensure equilibrium had been reached. All procedures of Murphy and colleagues were followed except that aliquots of 1.2 ml of 1:2 tissue weight:saline volume ratio rather than undiluted fat were used. Furthermore, each PC sample was counted once for 10 min and recounted the following day. Less than a 1% error in counts was achieved counting aliquots of approximately 10,000 dpm per sample. The quantity of MBP in each equilibration vial was 0.3 µg (
62,000 dpm). Tissue:PC and saline:PC partition coefficients were calculated using the methods of Murphy and colleagues. For blood, the amount of MBP in reference and test vials, AR and AT respectively, were expressed as
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Setting AR = AT and rearranging terms yielded the following equation for calculation of the blood:PC partition coefficient:
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Estimated partition coefficients.
The algorithm of Poulin and Krishnan (1995) was used to estimate the nonionized MBP fat:blood, testes:blood, and muscle:blood partition coefficients from the n-octanol:water partition coefficient (Ko/w). A Ko/w for nonionized MBP of 3.03 was estimated based on its chemical structure using ProLogP 2.0 software (CompuDrug Chemistry Ltd., Burlingame, CA) and used as an input to the Poulin and Krishnan algorithm. A testes water fraction of tissue weight of 0.80, a total lipids fraction of tissue weight of 0.023 (Fiserova-Bergerova, 1983), a phospholipid (fraction of total lipids) of 0.64 (Williams et al., 1945
), and a neutral lipids fraction of total lipids of 0.36 were used to calculate the testes:blood partition coefficient. The slowly perfused tissues:blood partition coefficient was set equal to the predicted muscle:blood partition coefficient, and the rapidly perfused tissues:blood partition coefficient was set equal to the predicted liver:blood partition coefficient.
For comparison to the measured partition coefficients, MBP tissue:blood partition coefficients were also estimated using the Poulin and Krishnan algorithm from the MBP Kow predicted at physiological pH (i.e., for ionized MBP) as calculated by ProLogD 2.0. This comparison was conducted to evaluate the predictive ability of the Poulin and Krishna algorithm.
Flow-limited PBPK model.
A flow-limited model structure for nonvolatile chemicals was adapted from Ramsey and Andersen (1984) to describe the dosimetry of DBP and MBP (Fig. 1A). In a flow-limited model, the rate of transport of chemical into the tissue is limited by blood flow to that particular tissue, whereas diffusion is assumed to be very rapid. Because intact DBP was measured in the blood after oral doses up to 857 mg/kg (NIEHS, 1994
; NIEHS, 1995
), the first-order oral absorption rate of DBP was set to zero, effectively removing all DBP compartments from the model except the small intestine. Because MBP is nonvolatile, the concentration of MBP in venous blood was assumed to be equal to the concentration of chemical in arterial blood; therefore no exchange of blood MBP with inhaled air was described. A testes compartment was separated from other rapidly perfused tissues because the testes are the target organ for reproductive toxicity of MBP in males. The blood was represented as a compartment as DBP, if given intravenously, is metabolized in the blood. The small intestine was included as a compartment rather than the whole gastrointestinal tract because pancreatic lipase accounts for most of the gut hydrolysis of DBP to MBP and is released directly into the small intestine via the pancreatic duct.
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Enterohepatic circulation PBPK model.
Enterohepatic circulation was considered a viable hypothesis because MBP is a charged molecule and conjugated MBP has a molecular weight above 325, which is considered a lower bound for molecular weight for enterohepatic circulation in the rat (Guthrie and Hodgson, 1987). Because MBP is conjugated in the rat and MEHP is not, MBP would be even more likely than MEHP to undergo enterohepatic circulation. Secondary peaks in MBP blood concentrations following intravenous exposure to MBP and oral exposure to DBP also indicated potential enterohepatic circulation (NIEHS, 1994
; NIEHS, 1995
). In addition, 10% of radiolabeled, intravenously administered [14C]DBP was recovered in the bile at 5 h, and 4.5% of an oral dose after 6 h, with both free and conjugated MBP identified in the bile (Kaneshima et al., 1978
). In another study, 44% of an orally administered DBP dose was recovered in the bile after 3 days (Tanaka et al., 1978
). Enterohepatic circulation of MBP was described in the model by adding a flow of MBP from the liver back to the small intestine with an adjustable time delay (Fig. 1B
). The time delay accounted for the time required for bile to flow from the liver to the intestinal lumen.
Diffusion-limited PBPK model.
Diffusion-limitation is also likely, as MBP is a relatively large charged molecule with a molecular weight of 222. In the diffusion-limited model, the rate of diffusion from the tissue blood into the tissue rather than blood flow limited the transport of MBP into the liver, fat, testes, and slowly and rapidly perfused tissues. Each of these compartments was further divided into extracellular (blood) and intracellular compartments (tissue), each with its own volume (Fig. 1C). The volume of the extracellular compartment was set to the tissue blood volume (Table 1
), and the volume of the intracellular compartment was set to the difference between the total tissue volume and the tissue blood volume. The permeation coefficient-surface area-cross product controlled the flow from the extracellular compartment to the intracellular compartment (See Appendix).
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Model simulation.
The resulting systems of mass-balance differential equations were solved by numerical integration using the Gear Algorithm for stiff systems by ACSLTOX (Advanced Continuous Simulation Language) (Pharsight, Mountain View, CA) on a Pentium II 300/mHz desktop PC. More details concerning the model equations can be found in the Appendix.
Model physiologic parameters.
The rat physiologic parameters, including tissue blood volumes, were taken from Model Parameters for PBPK Models (1994) with the exception of the testes blood flow rate (Tabrizchi and Pang, 1993), fat blood volume (Andersen et al., 1993
), and testes blood volume (Chubb and Desjardins, 1982
). Body weight was estimated to be 265 kg for an 11-week-old rat using an age-related growth equation for F-344 rats (ILSI, 1994
). Parameter values are summarized in Table 1
. Exposure-related parameters were set up according to conditions in each individual experiment.
Adjustable parameters.
Metabolism and absorption rates, permeation coefficient-surface area-cross product, enterohepatic circulation rate, and time delay were estimated from pharmacokinetic data in adult male rats by maximum likelihood parameter estimation as implemented in the ACSLopt software. The Nelder-Mead algorithm was used for likelihood maximization, and the heteroscedasticity parameter () was fixed at 2.0 for consistency between data sets. The heteroscedasticity parameter is a parameter in the power-of-the-mean error model used in ACSLopt. A heteroscedasticity parameter of 2.0 means that the standard deviation is proportional to the value of the mean (i.e., relative error). This value of 2.0 was appropriate since the variance in concentration measurements was usually proportional to the concentration value.
There were 46 adjustable parameters in each model. Initially, the adjustable parameters were optimized no more than three at a time to different data sets in the nested approach described below. For the flow-limited model, MBP blood concentrations following MBP intravenous exposure were used to estimate liver MBP metabolism parameters (VMmax,l and KMm,l). Blood concentrations following MBP oral exposure were then used to estimate the first-order absorption rate of MBP (kMa), holding metabolic parameters constant. Next, holding MBP metabolism and absorption parameters constant, MBP blood concentrations following oral exposure to 43, 50, 100, 200, or 857 mg/kg DBP were used to estimate the gut metabolism rate parameter (kDsi). Finally, an overall optimization that included all the data and parameters was conducted using the nested approach estimates as initial values. The heteroscedasticity parameter was allowed to vary from 2.0 for this final total optimization. Individual animal rather than mean concentrations were used to more accurately estimate
. This resulted in an optimal set of parameter estimates with an associated log-likelihood function value. Log-likelihood function plots were examined at each step and after the total optimization to validate that local optimal parameter estimates were found and to investigate covariance between parameters.
For each alternative model structure, the same approach to parameterization was followed. All adjustable parameters not found in the flow-limited model (e.g., the permeation surface area-cross product for the diffusion-limited model) were also initially estimated from the intravenous MBP blood data simultaneously with MBP metabolism parameters. Again, a full optimization of all parameters to all data was performed.
MBP time course data.
MBP plasma concentration time courses were obtained from two studies (NIEHS, 1994; NIEHS, 1995
). These studies included concentrations following an intravenous exposure of 11-week-old Wistar Furth rats to 8 mg/kg MBP, or an oral exposure of Wistar Furth rats to 34 mg/kg MBP, 43 mg/kg DBP, or 857.1 mg/kg DBP, with one rat at each time point (NIEHS, 1994
). Concentrations were also measured following oral exposure of 11-week-old Sprague-Dawley rats to 50, 100, or 200 mg/kg DBP with three rats at each time point (NIEHS, 1995
). Additional details of the experimental design and analytical techniques can be obtained from the respective study reports. The data used in the present study were taken directly from the study reports for each individual animal. DBP was not detected in any sample. The ratio of plasma MBP concentrations to whole blood MBP concentrations was predicted to be 1.1 at physiologic pH by the Poulin and Krishnan Ko/w algorithm; hence, we thought it reasonable to use plasma concentration time courses to fit model parameters for describing whole blood concentrations.
PBPK model discrimination.
The ability of each alternative model structure to fit the experimental data was compared directly to the flow-limited PBPK model using the log-likelihood ratio test. The log-likelihood ratio test could be used to discriminate between models because each alternative model could be reduced to the flow-limited model by removing 12 additional adjustable parameters and reidentifying some model parameters. The test statistic L was calculated as
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The combined pH trapping and diffusion-limited model was compared to the pH trapping model with df = 1. The test statistic L was compared to the critical value of the 2 statistic at the
= 0.05 significance level.
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RESULTS |
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The measured partition coefficients were used in simulations with the flow-limited, enterohepatic circulation, and diffusion-limited models. The partition coefficients for unionized MBP obtained with the Poulin and Krishnan algorithm were used for the pH trapping model and for the combined pH trapping-diffusion limitation model. The algorithm-derived values were used for these latter models, as MBP is largely ionized under the laboratory conditions used for partition coefficient determinations. Because laboratory measurement of the MBP muscle:blood partition coefficient did not produce a meaningful value (Table 2), this partition coefficient was set equal to the MEHP muscle:blood partition coefficient (0.38), which was measured using the same vial equilibration technique (Keys et al., 1999). The actual MBP muscle:blood partition coefficient would be expected to be less than the MEHP muscle:blood partition coefficient, as MBP is more hydrophilic than MEHP. Multiple optimizations were run at lower values of the muscle:blood partition coefficient (as low as 0.2) to test if the results of the model discrimination process were sensitive to this parameter. Order and significant differences of alternative models were unaffected by the choice of the muscle:blood partition coefficient within these ranges. The rapidly perfused tissue:blood partition coefficient was set equal to the liver:blood partition coefficient.
Simulations of Rat Blood Concentrations after Intravenous MBP Exposure
The flow-limited model provided a poor fit to the blood concentrations following intravenous exposure to 8 mg/kg MBP. The enterohepatic circulation model was statistically a better fit than the flow-limited model (Table 3). The enterohepatic circulation model underpredicted early time points. However, it accurately simulated the secondary peak concentration of MBP. The diffusion-limited model was also a statistically better fit than the flow-limited model (Table 3
) and successfully predicted the early MBP time points. The pH trapping model was also a statistically better fit than the flow-limited model; however, all MBP concentrations were underpredicted (Fig. 2
). A diffusion-limited, pH trapping model was a statistically better fit than the pH trapping model (Table 3
) and successfully predicted both early and late time points due to the addition of diffusion-limitation (Fig. 2
).
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Adjustable Parameter Estimates
The estimated values of the adjustable parameters in the diffusion-limited, pH trapping model are presented in Table 4. Two dimensional log-likelihood function surface plots were examined for all 10 combinations of pairs of parameters. A distinct optimum was found for each pair with the exception of VMmax,l and KMm,l, where a ridge indicating covariance between parameters was found. Thus estimates for VMmax,l and KMm,l must be considered to be preliminary. Blood concentrations following various doses of MBP intravenously would result in unique estimates of these two parameters.
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DISCUSSION |
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In vitro tissue:blood partition coefficients for MBP were smaller than those measured for MEHP in all tissues except the testes, whereas MBP was an order of magnitude more soluble in saline than MEHP. Tissue:blood partition coefficients were expected to be smaller for MBP than MEHP, as MBP has a shorter side/chain length and is thus less lipophilic than MEHP. Gut metabolism of DBP to MBP was modeled as a linear process, whereas saturable metabolism was necessary to model DEHP gut metabolism (Keys et al., 1999). This difference is probably due to the higher doses of DEHP than DBP in the oral pharmacokinetic study, which was used for estimation of gut metabolism parameters (2000 mg/kg DEHP vs. 43857 mg/kg DBP). The fact that DBP metabolism could be modeled as a linear process does indicate, however, that the gut metabolism of phthalate diesters is not saturated until very high concentrations in the rat. Maximum likelihood estimates of a MEHP Vmax value in the liver were similar to those for MBP, 12.0 mg/h and 11.6 mg/h, respectively, whereas estimated MEHP Km value in the liver was 1.5 orders of magnitude lower than MBP, 0.5 mg/L and 18.2 mg/L, respectively. The fact that the MEHP Km was estimated to be lower than the MBP Km may indicate phthalate ester metabolizing P450s have a higher affinity for MEHP than MBP. However, as covariance of Vmax and Km in the liver prevented the unique estimation of their values, more in vivo or in vitro data are necessary before this can be confirmed. Also, the metabolism of MBP in the liver, which is currently represented as one saturable rate, is really a composite of at least two metabolic pathways and urinary excretion. The estimated rate of MBP absorption was slightly higher than for MEHP, 9.9 h-1 versus 7 h-1, which agrees with the results of a previous study in which investigators found that the amount of phthalate ester flux was 9.6 µmol/h for MBP and 7.7 µmol/h for MEHP in an everted gut-sac preparation (White et al., 1980).
NIEHS (1994) and NIEHS (1995) used Wistar-Furth and Sprague-Dawley rats, respectively. Our analyses of blood MBP after oral dosing with DBP examined data from both of these studies without attempting to develop rat strain-specific PBPK models. No data are currently available that would support such model development. Moreover, reasonable fits to all of the data were obtained with a single model, suggesting that intrastrain differences in the pharmacokinetic behavior of DBP and MBP are not large.
The results of this work show that a diffusion-limited, pH trapping PBPK model can accurately describe the in vivo pharmacokinetics of di(n-butyl) phthalate and its metabolite mono(n-butyl phthalate). Consideration of tissue pH gradients and ionized monoester along with diffusion-limitation is critical for description of MBP blood and thus target organ dosimetry. The enterohepatic circulation model underestimated MBP peak blood concentrations following oral exposure to DBP, but this does not mean that enterohepatic circulation is not occurring. In fact, the existence of secondary peaks in both the iv and oral concentration time courses and the measurement of MBP in the bile suggests that enterohepatic circulation most likely is occurring. The results of this work do, however, indicate that enterohepatic circulation is not critical for a reasonable overall description of MBP blood dosimetry. Enterohepatic circulation may affect only the variance in the concentration-time curves (nonmonotonic elimination phase) but not the AUC and peak concentration values. The AUC and peak concentrations are the most crucial to estimate, as they are the most likely candidates for a dose metric for phthalate monoester.
The adequate and superior fit of the diffusion-limited, pH trapping model for DBP and MBP combined with the adequate and superior fit of the pH trapping model for DEHP and MEHP are initial steps toward developing a generic phthalate ester PBPK model. The next steps toward developing a generic model are validation of the DBP and MBP PBPK model and application of the current model structure to other phthalate esters. Development of a generic phthalate ester PBPK model would allow for more biologically based human risk assessments of male reproductive toxicity for exposure to one or more phthalate esters. Once parameterized for phthalate-specific parameters, target tissue doses in testes could be predicted to help interpret rat toxicological studies. Also, the model could be scaled to humans by incorporating human data on absorption and urinary excretion that will allow predictions of target tissue doses in humans. This would decrease the level of uncertainty in the current risk assessments for phthalate esters. In addition, concern for the effects of phthalates following in utero exposure has been raised as recent work that shows this to be the most sensitive end point (Mylchreest et al., 1998; NTP, 1991
; Wine et al., 1997
). A generic phthalate ester PBPK model could be extended to predict fetal concentrations of diester and monoester and thereby aid in decreasing uncertainty in risk assessments for pregnant women exposed to one or more phthalate esters.
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APPENDIX |
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Flow-Limited Model
The flow-limited model equations for DBP and MBP were identical to those used by Ramsey and Anderson (1984) with the following exceptions.
The rates of change of the amount of DBP and MBP in the small intestine are given by
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The rate of change of MBP in the blood is given by
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Enterohepatic Circulation Model
The enterohepatic circulation model included two additional parameters: ken, the first-order rate of transfer of MBP from the liver to the small intestine via the bile (h-1) and , a time delay (h). The rate of change of the concentration of MBP in the small intestine is given by
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Diffusion-Limited Model
The diffusion-limited model included one additional parameter not found in the flow-limited model: PA, the permeation coefficient-surface area-cross product (h-1). The rate of change of the amount of MBP in the blood and tissue fractions is expressed as
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pH Trapping Model
The pH trapping model introduces three additional parameters: pHE is the extracellular pH; pHI, the intracellular pH, and the MBP pKa. The concentrations of nonionized (Ci,Ea) and total MBP (Ci,E) in extracellular tissue i (i = l, f, t, sp, or r) are derived from the Henderson-Hasselbalch equations and are expressed as
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The concentration of nonionized (Ci,Ia) is reached by instantaneous partitioning between unionized MBP in tissue and blood and is given by
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Diffusion-Limited pH Trapping Model
The diffusion-limited, pH trapping model combines the equations and parameters of the diffusion-limited and pH trapping models. The rates of change of the total amount of MBP in the extracellular and intracellular compartments of tissue i (i = l, f, t, sp, or r) are given by
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Approximate Partition Coefficient Standard Error Calculation
The following equations were derived based on Taylor's series expansions to calculate approximate standard error of mean tissue:propylene partition coefficients (s\&lgr;t:PC)
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ACKNOWLEDGMENTS |
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NOTES |
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