Disposition of Radioactivity in Fischer 344 Rats after Single and Multiple Inhalation Exposure To

The retention, distribution, metabolism, and excretion of [C]octamethylcyclotetrasiloxane (D4) were studied in Fischer 344 rats after single and multiple exposures to 7, 70, or 700 ppm [C]D4. Subset groups were established for body burden, distribution, and elimination. Retention of inhaled D4 was relatively low (5–6% of inhaled D4). Radioactivity derived from [ C]D4 inhalation was widely distributed to tissues of the rat. Maximum concentrations of radioactivity in plasma and tissues (except fat) occurred at the end of exposure and up to 3 h postexposure. Maximum concentrations of radioactivity in fat occurred as late as 24 h postexposure. Fat was a depot, elimination of radioactivity from this tissue was much slower than from plasma and other tissues. With minor exceptions, there were no consistent gender effects on the distribution of radioactivity and the concentrations of radioactivity were nearly proportional to exposure concentration over the exposure range. Excretion of radioactivity was via exhaled breath and urine, and, to a much lesser extent, feces. Urinary metabolites included dimethylsilanediol and methylsilanetriol plus five minor metabolites. Relative abundance of these metabolites was the same from every test group. Elimination was rapid during the first 24 h after exposure and was slower thereafter (measured up to 168 h postexposure). In singly-exposed female (but not male) rats, small dosedependent shifts in elimination pathways were seen. After multiple exposures, the elimination pathways were doseand gender-independent. These data define possible pathways for metabolism of D4 and allow estimation of the persistence of D4 and/or its metabolites in rats. Octamethylcyclotetrasiloxane (D4) 3 is a clear, odorless, silicone fluid of molecular weight 296 with alternating silicon-oxygen bonds connected in a ring (cyclic) arrangement with two methyl groups covalently bonded to each silicon atom (-(CH3)2SiO-) (Fig. 1). D4 is an intermediate in the industrial manufacture of polydimethylsiloxane, a silicone polymer that is used widely in industrial and consumer applications (Stark et al., 1982). D4 is also an ingredient in selected personal care products, including antiperspirants and skin care products. In addition, workplace exposures occur in the production of D4 and other silicone materials via the respiratory route. The chemical and physical properties of D4 are well known (Varaprath et al., 1996), and the distribution and persistence of mixtures of low molecular weight silicones after s.c. injections in mice were reported by Kala et al. (1998). However, there is little published data on the biological fate and toxicological effects of D4 after relevant routes for human exposure. McKim et al. (1998) reported that repeated inhalation exposure to high concentrations of D4 produces a reversible and dose-related liver enlargement with significant induction of cytochrome P-450 CYP2B1/2. In addition, the enzyme induction profile produced during exposure to D4 was comparable to that observed with phenobarbital (PB). The results of these studies provided compelling evidence that D4 was a PB-like inducer of hepatic microsomal enzymes in the Fischer 344 rat. Preliminary results presented in abstract form demonstrated that pretreatment of female rats with PB before administration of D4 increased the excretion rate and metabolism of D4 (Salyers et al., 1996). Thus enzyme induction, either by D4 itself or by other exogenous chemicals, may influence disposition of D4 in the rat. Because of the widespread use of D4 and the potential for human exposure, a comprehensive program has been initiated to assess the kinetics, metabolism, induction, and toxicity of D4 in rats after relevant routes of exposure. In addition, studies have been initiated to examine the pharmacokinetics of D4 in humans (Utell et al., 1998). Specific objectives of these studies were to assess the effects of dose and gender on the distribution, persistence, and pathways for elimination of D4 and its radioactive metabolites after either a single inhalation exposure to [C]D4 or multiple inhalation exposures to unlabeled D4 followed by a single inhalation exposure to [ C]D4. These studies provide information that may be helpful in characterizing the risk of human populations exposed to D4. Supported in part by the Silicones Environmental, Health and Safety Council of North America. 1 Present address: Lorus Therapeutics, Inc., 7100 Woodbine Ave., Suite 215, Markham, ON Canada L3R 5J2. 2 Present address: Sierra Biomedical Inc., 587 Dunn Circle, Sparks, Nevada. 3 Abbreviations used are: D4, octamethylcyclotetrasiloxane; AUC, area under the concentration curve versus time; t1/2, apparent elimination half-life; tmax, time to maximum concentration; Cmax, maximum concentration; PB, phenobarbital; GC/MS, gas chromatography mass-spectrometry; 2-EE, 2-ethoxyethanol. Send reprint requests to: Dr. Kathleen P. Plotzke, Manager, Toxicology Research, Health and Environmental Sciences, Dow Corning Corporation, Mail no. CO3101, 2200 W. Salzburg Rd., Midland, MI 48686. E-mail: kathy.plotzke@ dowcorning.com 0090-9556/00/2802-0192–204$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 2 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 192 at A PE T Jornals on Sptem er 7, 2017 dm d.aspurnals.org D ow nladed from Experimental Procedures Test Animals. Young adult male and female Fischer 344 rats weighing approximately 125 to 210 g were obtained from Charles River Canada Inc. (St. Constant, Quebec, Canada). Animals were housed individually in suspended stainless steel, wire mesh bottom cages in a room designed to be maintained at 65–78°F, and 30 to 70% relative humidity. A commercial diet (PMI Certified Rodent Chow, 5002; PMI Feeds Inc., St. Louis, MO) and municipal drinking water were available ad libitum except during exposure periods. The photoperiod alternated 12 h of light with 12 h of darkness. During inhalation exposures, animals were housed in polycarbonate restrainers to achieve noseonly exposure. After exposure, animals allocated to the elimination components of the experiments were individually housed in Roth-type glass metabolism cages designed for the separate collection of urine, feces, and expired air. These animals were acclimated to the metabolism caging for 48 h before being placed on study. Airflow through the metabolism caging was kept at a minimum of 500 ml/min. Test Chemicals. D4 (CAS 000556672) was obtained from Dow Corning Corporation (Midland, MI). The purity was determined to be 99.8% by gas chromatography-mass spectrometry (GC/MS). [C]Labeled D4 was obtained from Wizard Laboratories (West Sacramento, CA). Radiochemical purity was confirmed by HPLC equipped with a radiometric flow detector. All dosing solutions had a radiochemical purity of 98.5% or greater. [C]Dose Solution Preparation and Analysis. [C]Dose solutions were prepared by diluting [C]D4 with unlabeled D4 to a target specific activity that allowed delivery of approximately 40 mCi of radioactivity to each animal over the 6-h exposure period. Specific activity was confirmed by liquid scintillation counting. Administration of Test Material. Animals were exposed to D4 vapor in a cylindrical flow-past nose-only inhalation chamber. All animals were conditioned to the exposure apparatus for 4 days before the start of the experiments. Targeted conditioning periods were 1, 2, 4, and 6 h on days 1 to 4, respectively. In single exposure studies, animals received one exposure to [C]D4 immediately after the conditioning period. In multiple exposure studies, after receiving the same conditioning, animals were subjected to fourteen 6-h exposures to unlabeled D4 followed on the 15th day by a 6-h exposure to [ C]D4. The test atmosphere was generated using a heated, flow-through vapor generator. Liquid D4 was fed using a peristaltic pump onto a column of glass beads wrapped with heating tape and kept at constant temperature with a rheostat. The D4 vapor was carried from the vapor generator into the inhalation chamber by a regulated airflow that entered the vapor generator below the glass beads. Chamber vapor concentration was controlled by adjusting the input D4 and airflow rates into the generator. Chamber airflow was set at a level determined to provide test atmosphere to each animal at a rate of approximately 500 ml/min. During animal exposures, samples of the test atmospheres were collected at approximately 15-min intervals from a representative animal breathing port and were analyzed for D4 by gas chromatography. Test atmosphere was also continually monitored throughout the exposure period by a Miran 1A infrared gas analyzer (The Foxboro Company, Foxboro, MA). During exposures, test atmosphere temperature, relative humidity, and oxygen concentration were recorded at a control breathing port at 15-min intervals. Chamber airflow was continuously monitored throughout each exposure. The achieved [C]D4 exposures (expressed as total mCi) during the 6-h interval (A) were calculated by the following equation: A(mCi) 5 B 3 C 3 D 3 E where: B 5 respiratory minute volume (VE, liters/min) calculated on the basis of body weight: VE 5 2.1 (BW grams) 0.75 (Guyton, 1947), C 5 exposure duration (360 min), D 5 actual [C]D4 chamber concentration (milligrams per liter): calculated on the basis of mean chamber [C]D4 concentration (ppm) measured by gas chromatography, the molecular weight of D4 (296.62 g/mol), and the molar gas volume corrected for pressure and temperature (24.060 liters/mol), and E 5 specific activity of [C]D4 (mCi/mg) determined on the liquid used to generate the chamber atmosphere. Experimental Design. Rats were weighed and assigned to subsets within each single or multiple exposure dose group by a computer-based stratified randomization procedure. Subset groups were established for body burden, distribution, and elimination components of the study, with four or five male or female animals per subset group, per dose. For both single exposure and multiple exposure studies, one male and one female animal per dose group were selected randomly to assess background radioactivity. Sample Collection and Storage. Body burden subset. For the single exposure studies, rats were removed from the exposure chamber, removed from the exposure restraint tube, injected i.p. with sodium pentobarbital, and euthanized by cervical dislocation approximately 3 to 5 min after removal from the inhalation chamber (when the animals appeared to have stopped breathing). The carcass, along with urine and feces collected from the exposure tube, were then immediately solubilized together. In the multiple exposure studies, to minimize any loss of radioactivity in the period between removal from the exposure chamber and the cessation of breathing, body burden animals were anesthetized by i.v. (tail vein) or i.p. injection of sodium pentobarbital and allowed to stop breathing before they were removed from the inhalation chamber. Thereafter the animals were processed as described above. Distribution subset. Rats were injected with sodium pentobarbital and euthanized by exsanguination from the abdominal aorta for collection of blood and tissues at 0, 1, 2, 3, 12, 24, 48, 72, 96, 120, and 168 h postexposure. Blood samples were transferred to heparinized tubes, mixed, and cooled on ice for approximately 10 min. Aliquots were transferred to scintillation vials and the remaining blood was centrifuged to prepare plasma. Blood and plasma were processed immediately for radioactivity measurement. In addition, various tissues (liver, lungs, perirenal fat, ovaries, vagina, and testes) were excised, weighed, and processed for radioactivity measurements as described below. Elimination subset. Immediately after exposure, rats were placed in Rothtype glass metabolism cages for collection of excreta (urine and feces) and expired air (CO2 and other volatiles). Excreta deposited in the exposure tube of each animal were collected at the end of exposure and counted separately. Urine and feces (collected over dry ice) and expired CO2 (trapped in a single 4 N KOH trap) were collected at 6, 12, and 24 h, and subsequently at 24-h intervals up to 168 h postexposure. Other expired volatiles were collected in two 2-ethoxyethanol (2-EE) traps (the first maintained on ice, the second on dry ice) at 1, 2, 4, 6, 9, 12, and 24 h, and at 24-h intervals up to 168 h 4 Additional tissues were collected and processed for radioactivity in a pilot study as well as the two studies presented here. In the interest of space, only data from select tissues will be discussed. Copies of the full reports can be obtained from the Environmental Protection Agency (EPA). Please refer to the Document Control Number (DCN) for the respective report. A Pilot Study for the Determination of C-Octamethylcyclotetrasiloxane (D4) Pharmacokinetics in Fischer 344 Rats following a Single Nose-Only Vapor Inhalation Exposure to 700 ppm C-D4, EPA DCN 86960000517. Pharmacokinetics of C-Octamethylycyclotetrasiloxane (D4) in the Rat following Single Nose-Only Vapor Inhalation Exposure, DCN 86960000024. Pharmacokinetics of C-Octamethylcyclotetrasiloxane in the Rat following 14 Repeat Daily Nose-Only Vapor Inhalation Exposures to Unlabeled D4 and a Single (Day 15) Exposure to C-D4 at Two Exposure Levels, EPA DCN 86970000875. FIG. 1. Chemical structure of D4. 193 DISPOSITION OF [C]OCTAMETHYLCYCLOTETRASILOXANE at A PE T Jornals on Sptem er 7, 2017 dm d.aspurnals.org D ow nladed from postexposure. Cage rinses (70% ethanol/water followed by distilled water) were performed at 72, 120, and 168 h postexposure. After 168 h of excreta collection, the animals were anesthetized by pentobarbital injection and subsequently euthanized by exsanguination. Whole blood, plasma, and tissues including the remaining carcass were collected as in the distribution group. To facilitate comparisons of the different routes of elimination in different dose/ gender groups, data from the metabolism cages were normalized. In each case the percentages of radioactivity eliminated by a particular route were calculated relative to the total radioactivity recovered in that particular elimination