Further comments on the bioavailability of [D.sub.4]. (Correspondence).

We would like to comment on a paper by Luu and Hutter (1) published in the November 2001 issue of EHP. We have developed multidose route, multi-species PBPK models for [D.sub.4] over the past several years. Our PBPK models have been presented in abstract form at several national meetings, and the complete inhalation model for [D.sub.4] in the rat was published earlier this year (2). In their paper, Luu and Hutter (1) incorrectly attribute several conclusions to our earlier abstracts, including the comment that our model did not describe blood concentrations during and after exposure. Surprisingly, they did not cite conclusions from our complete, peer-reviewed documentation of our model. We would like to point out some important differences between their model and our [D.sub.4] model. We would like to address several issues: a) the unconventional model structure and inappropriate use of available pharmacokinetic data to estimate the blood:air partition coefficient by Luu and Hutter (1); b) the process by which all available pharmacokinetic data should have been used to ensure adequate validation of their PBPK model; and c) the unusual kinetic behavior of [D.sub.4] compared to other volatile organic compounds that needs to be captured in any kinetic model for this compound.

A major difference in Luu and Hutter's model (1) and our published model (2) is the value used for the blood:air partition coefficient ([Pb.sub.:a]). Our estimate of [P.sub.b:a] derived from the measured concentrations of parent [D.sub.4] in blood at the end of a 6-hr exposure was 0.8; our direct measurements of the [P.sub.b:a] by equilibration of [D.sub.4] between blood and air in vitro gave a value near 4.0. Luu and Hutter used a much higher value of 20 and reported that they were able to describe both the rat and human inhalation results. It is of interest to determine why there would be such a large discrepancy in a critical parameter between the two models.

Luu and Hutter's model for [D.sub.4] in the rat (1) is based on studies in which total radioactivity was measured in blood after exposure of rats to [sup.14]C-[D.sub.4]. Luu and Hutter (1) used the radioactivity data from Plotzke et al. (3) and assumed that the radioactivity in blood was parent compound. In our work, we modeled parent [D.sub.4] and metabolites separately. By the end of the 6-hr inhalation exposure in rats, the majority of radioactivity in blood is metabolite (about a 3- to 4-fold greater concentration of metabolite vs. parent [D.sub.4] at the end of the exposure). After the 6-hr exposure, [D.sub.4] is rapidly eliminated by exhalation compared to the metabolites, and the discrepancy between total radioactivity and parent [D.sub.4] only increases. To predict these artificially high blood levels and retain these high concentrations for long periods of time, Luu and Hutter's model requires an artificially high estimate of the partition coefficient, thus the use of 20 in their model versus 1.0 in our model in which parent [D.sub.4] and metabolites were described separately. Luu and Hutter (1) then scaled the model with the high partition coefficient to humans. In this case the data in their paper was for parent [D.sub.4]; nonetheless, they still showed good correspondence between data and model predictions. We believe that this agreement is quite misleading and related to differences between their human modeling approach and conventional approaches used with other volatiles. Their ability to fit the human [D.sub.4] was based on an artificial constraint added to limit retention of inhaled [D.sub.4].

Based on the equations of Ramsey and Andersen (4), a paper cited as the basis of Luu and Hutter's work, the concentration of styrene in the arterial air (Cart) could be approximated from a steady-state formula published by Andersen (5):

[1] [C.sub.art] = [P.sub.b:a] X [Q.sub.alv] x [C.sub.inh] / [Q.sub.alv] + [P.sub.b:a] X [E.sub.H] x [Q.sub.H]

where [Q.sub.alv] is the alveolar ventilation, [E.sub.H] is hepatic extraction, [Q.sub.H] is the hepatic blood flow, and [C.sub.inh] is the inhaled concentration of compound. In PBPK models, inputs include partition coefficients, inhaled concentrations, and the suite of physiologic factors, including blood flows, breathing rates, and characteristics of metabolizing tissues. Using all of these factors together, it is possible to predict the amount of inhaled compound that is retained during respiration. For modeling exposures in rats, Luu and Hutter (1) correctly used the ventilation x the inhaled concentration as the input term to the arterial blood in the rats. In contrast, for the human modeling Luu and Hutter (1) cited the differences (input-output) measured in a human study from the University of Rochester (6) and applied them as a constraint on the model. Thus, their input is ([Q.sub.alv] x [C.sub.inh] X proportion retained). Because the proportion retained was only 0.1, the model required an anomalously high blood:air partition coefficient to achieve blood concentrations equal to the inhaled air concentrations. (This behavior follows from Equation 1 if the proportion retained is included empirically.) Our PBPK model for [D.sub.4], following previous approaches with volatile compounds such as styrene, describes parent [D.sub.4] concentrations in rat and humans without artificial constraints on uptake. The proportion retained is an output of the model, not a constraint. In this fashion, both rat and human uptake curves are adequately described in our modeling efforts with [P.sub.b:a] = 1.0.

The novel kinetic behavior referenced in the title of our paper (2) is the persistence of nonexchangeable [D.sub.4] in blood at long times after exposure. We only identified the necessity to include this bound form in blood because of our efforts to fit blood and exhaled [D.sub.4] during both the exposure and the postexposure periods. Luu and Hutter's model (1) also included blood sequestration from the plasma pool of [D.sub.4]. (The equation in their paper for the weakly bound compartment appears to be incorrect. The last term in their paper for this equation should be [k.sub.si] x [C.sub.wk] rather than [k.sub.si] X [C.sub.str]. According to the author's description

[V.sub.weak] [d[C.sub.wk] / dt = [k.sub.wi][C.sub.ai] + [k.sub.so][C.sub.str] + [k.sub.wo][C.sub.wk] + [k.sub.si][C.sub.str],

where [C.sub.ai] is the concentration of [D.sub.4] dissolved in plasma; [C.sub.wk] is the concentration of [D.sub.4] weakly protein bound in plasma; [C.sub.str] is the concentration of [D.sub.4] strongly protein bound in plasma; [k.sub.wi] is forward rate constant for weak protein binding of [D.sub.4] in plasma; [k.sub.si] is forward rate constant for strong protein binding of [D.sub.4] in plasma; [k.sub.so] is reverse rate constant for strong protein binding of [D.sub.4] in plasma; [k.sub.wo] is reverse rate constant for weak protein binding of [D.sub.4] in plasma; [V.sub.weak] is the volume of weakly bound plasma.

Another similarity in structure of the two models is the use of multiple fat compartments. Luu and Hutter (1) used a diffusional movement from a single fat compartment into a sequestered compartment within the main fat compartment. In our model, we described different fat compartments within the body with different time constants for equilibration. Luu and Hutter (1) referred to blood flow to deep fat, although the description and equations indicate a diffusional movement from weakly bound fat to the deep fat compartment. Their equation for the deep fat compartment is also inaccurate as written; it should show a term for movement from the weakly bound fat compartment. In its present form in their paper (1), the rate of change of mass for the deep fat would always be zero. [The equation for the lung compartment in Luu and Hutter's paper (1) also has an error, with [C.sub.lung] appearing twice in the second term or me mass balance equation.]

The model structure used by Luu and Hutter (1) for intravenous dosing actually is for intra-arterial dosing, in which the compound is placed in the arterial blood and infused into tissues rather than introduced into the venous blood, where it must traverse the lung with opportunity for exhalation before passing to the arterial blood. For a compound with a low [P.sub.b:a] such as [D.sub.4], it is important to have physiologic realism in the dosing route in order to estimate exhaled [D.sub.4] accurately after intravenous dosing.

Another issue is that Luu and Hutter (1) should have used all available pharmacokinetic data to insure adequate validation of their PBPK model. After configuring the model for intravenous dosing, a practice common to many pharmacokinetic studies, Luu and Hutter (1) predicted plasma and fat concentrations for a single inhalation exposure of rats to [D.sub.4]. The model overestimated the early time points in fat. In addition, the overall time course in plasma was underestimated for this one attempt at extrapolation and validation. Surprisingly, this validation exercise used a single study from an extremely rich data set on the inhalation pharmacokinetics of [D.sub.4] in rats. The data used for dose route extrapolation and validation once again were for radioactivity rather than for parent [D.sub.4] in blood and fat, whereas their pharmacokinetic model was purportedly for parent [D.sub.4] alone.

Plotzke et al. (3) performed pharmacokinetic studies of inhaled [D.sub.4] in male and female rats at three exposure concentrations for both single and multiple exposures. These inhalation studies generated important data on. tissue time courses of [D.sub.4] in a large set of tissues, as well as in exhaled breath concentrations. Similarly, the available human data for interspecies extrapolation include exhaled breath concentrations and blood concentrations from volunteers (6). Any model validation exercise should consider all available kinetic information and not rely on a limited selection of these results. Luu and Hutter's (1) conclusions regarding validation should be regarded as preliminary until their PBPK model is rigorously tested against more complete data sets. For Luu and Hutter to assert that prediction of a limited set of available human data from an unconventional model for inhalation constitutes dose-route and interspecies validation of their PBPK model is an over-interpretation of available information.

A third area of concern in Luu and Hutter's study (1) involves the unusual kinetic characteristics of [D.sub.4]. There is little doubt that the defining characteristic of [D.sub.4] is its lipophilicity, including a high fat:blood partition coefficient [(P.sub.f]). We determined by vial equilibration methods that [P.sub.f] was 500-600 in rats (2). The overall kinetic behavior of [D.sub.4], however, is related to several important characteristics: lipophilicity, high metabolic clearance from liver, and high exhalation clearance due to its relatively low [P.sub.b:a]. This suite of characteristics insures that [D.sub.4] does not bioaccumulate excessively with repeated dosing. Although both Luu and Hutter's model (1) and our PBPK model agree that the fat-time constant is of the order of several weeks, [D.sub.4] behaves much differently from poorly metabolized, nonvolatile compounds that bioaccumulate extensively with multiple exposures. The blood levels of [D.sub.4] do not increase with daily exposures and the fat concentration increases only slightly, as noted in the multiple exposure studies reported by Dow Corning scientists and analyzed with our more complete PBPK model (2). On a fairly minor note, the pharmacokinetic models developed by both groups are linear, low-dose models. Luu and Hutter (1) called the kinetics of the intravenous administration nonlinear. The appropriate terminology would be polyexponential, not nonlinear.

We are pleased to see PBPK modeling approaches for evaluating interspecies differences in disposition appear in EHP; however, Luu and Hutter's statements regarding our inability to model postexposure [D.sub.4] levels are inaccurate. The postexposure kinetic behavior of [D.sub.4] is determined by a combination of free [D.sub.4] and [D.sub.4] in a nonexchangeable compartment. These time-course curves have been accurately described at various concentrations after both single and multiple exposures in male and female rats with our PBPK model structure (2). As Luu and Hutter noted, we did not report extrapolation to humans. The reason for this was that we were in the process of completing a more definitive examination of human inhalation kinetics from two complete human data sets on a total of 18 exposures. These analyses have now been completed (7,8).

To summarize our human modeling, we found that the structure of the rat PBPK model for [D.sub.4] with a [P.sub.b:a] of near 1.0, when scaled appropriately, was entirely adequate for describing all available data from human volunteers. We are concerned about the inaccurate attribution of conclusions of our modeling efforts by Luu and Hutter (1) and appreciate the opportunity to provide clarification on these points. We emphasize that the kinetics of [D.sub.4] are well described with [P.sub.b:a] = 1.0 in both rats and humans, when sequestration in blood lipids is included in the model structure. Because of the high rate of metabolism and exhalation of poorly soluble [D.sub.4] from blood, there should be little tendency for [D.sub.4] to bioaccumulate in any tissues upon repeated exposures.

REFERENCES AND NOTES

(1.) Luu H-MD, Hutter JC. Bioavailability of octamethylcyclotetrasiloxane ([D.sub.4]) after exposure to silicones by inhalation and implantation. Environ Health Perspect 109:1095-1101 (2001).

(2.) Andersen ME, Sarangapani R, Reitz RH, Gallavan RH, Dobrev ID, Plotzke KP. Physiological modeling reveals novel pharmacokinetic behavior of inhaled octamethylcyclotetrasiloxane in rats. Toxicol Sci 60:214-231 (2001).

(3.) Plotzke K, Crofoot S, Ferdinandi E, Beattie J, Reitz R, McNett D, Meeks R. Disposition of radioactivity in Fischer 344 rats after single and multiple inhalation exposure to 14C- Octamethylcyclotetrasiloxane--[D.sub.4]. Drug Metab Dispos 28:192-204 (2000).

(4.) Ramsey JR, Andersen ME. A physiologically based description of the inhalation pharmacokinetics of styrene in rats and humans. Toxicol Appl Pharmacol 73:159-175 (1984).

(5.) Andersen ME. A physiologically based toxicokinetic description of the metabolism of inhaled gases and vapors: analysis at steady state. Toxicol Appl Pharmacol 60:509-526 (1981).

(6.) Utell MJ, Gelein R, Yu CP, Kenaga C, Geigel E, Torres A, Chalupa D, Gibb FR, Speers DM, Mast RW, Morrow PE. Quantitative exposure of humans to an octamethylcyclotetrasiloxane (D-4) vapor. Toxicol Sci 44(2):206-213 (1998).

(7.) Reddy MB, Dobrev ID, Utell MJ, Morrow PE, Plotzke KP, Andersen ME. Human inhalation pharmacokinetics of octamethylcyclotetrasiloxane (D4): evaluation with a physiological model [Abstract 241]. Toxicologist 66:50 (2002).

(8.) Reddy MB, Andersen ME, Morrow PE, Dobrev ID, Varaprath S, Plotzke KP, Utell MJ. Unpublished data.

Melvin E. Andersen
Ivan D. Dobrev
Micaela B. Reddy
Colorado State University
Foothills Campus
Fort Collins, Colorado
E-mail: [e-mail]

Ramesh Sarangapani
ICF Consulting
Research Triangle Park, North Carolina

Richard H. Reitz
RHR Toxicology Consulting Services
Midland, Michigan

Kathleen P. Plotzke
Dow Corning Corporation
Midland, Michigan