Potential estrogenic and antiestrogenic activity of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) in immature rats using the uterotrophic assay.

The cyclic siloxane octamethylcyclotetrasiloxane (D4) and the linear siloxane hexamethyldisiloxane (HMDS) have numerous industrial and consumer applications and thus have the potential for human exposure. The present study was undertaken to examine potential estrogenic and antiestrogenic activities of D4 and HMDS. To address potential differences in sensitivity between rat strains the study used both Sprague-Dawley (SD) and Fischer 344 (F-344) rats. Estrogenicity of the test compounds was determined by measuring absolute and relative uterine weights in immature rats and by monitoring uterine epithelial cell height. In order to place the data obtained for D4 into perspective relative to strong and weak estrogenic compounds, the response produced by D4 at 0, 10, 50, 100, 250, 500, and 1000 mg/kg/day was compared to responses produced by ethinyl estradiol (EE) (1, 3, 10, or 30 microg/kg/day), diethylstilbestrol dipropionate (DES-DP) (0.5, 1.5, 5, 15 microg/kg/day), and coumestrol (CE) (10, 35, 75, 150 mg/kg/day). Antiestrogenic effects were evaluated by co-administering D4 (500 mg/kg/day) with EE at 1, 3, 10, and 30 microg /kg/day. All compounds were administered in sesame oil at a volume of 5 mL/kg by oral gavage. Beginning on postnatal day 18 (SD) or 21 (F-344) each pup (12 per group) received a single dose of test compound once a day for 4 consecutive days. The pups were euthanized the morning after the last treatment and their uteri removed, weighed, and processed for histological examination. EE and DES-DP produced a significant dose-dependent increase in absolute and relative uterine weights and uterine cell height. The maximum increase in uterine weight following EE exposure was approximately 350% relative to controls in both strains. The weak phytoestrogen CE also produced a dose-related increase in absolute and relative uterine weight and epithelial cell height, but the response occurred over a much higher range of doses. At the highest dose of CE, uterine weight was increased approximately 230% relative to controls. Following exposure to D4, absolute and relative uterine weights and uterine epithelial cell height were statistically significantly increased in both strains of rats at doses above 100 mg/kg/day. In terms of uterine weight, D4 was approximately 0.6 million times less potent than EE or DES-DP in SD pups and 3.8 million times less potent than EE or DES-DP in F-344 pups. The maximal increase in uterine weight, relative to controls, produced by D4 at 1000 mg/kg/day was approximately 160% in SD rats, while the maximum increase produced by D4 in F-344 rats was 86%. D4 co-administered over a wide range of EE doses, resulted in a significant reduction in uterine weight compared to EE alone. HMDS was evaluated in SD rats only. The response produced by HMDS (600 and 1200 mg/kg/day) was compared to EE (3 microg/kg/day). Antiestrogenic effects were evaluated by co-administering HMDS (1200 mg/kg/day) with EE at 3 microg/kg/day. HMDS had no measurable effect on uterine weight under the experimental conditions described here. However, HMDS coadministered with EE did produce a small, but statistically significant reduction in uterine weight compared to EE alone. In conclusion, D4 showed weak estrogenic and antiestrogenic activity that was several orders of magnitude less potent than EE, and many times less potent than the weak phytoestrogen CE.

Physiological modeling reveals novel pharmacokinetic behavior for inhaled octamethylcyclotetrasiloxane in rats.

Octamethylcyclotetrasiloxane (D4) is an ingredient in selected consumer and precision cleaning products. Workplace inhalation exposures may occur in some D4 production operations. In this study, we analyzed tissue, plasma, and excreta time-course data following D4 inhalation in Fischer 344 rats (K. Plotzke et al., 2000, Drug Metab. Dispos. 28, 192-204) to assess the degree to which the disposition of D4 is similar to or different from that of volatile hydrocarbons that lack silicone substitution. We first applied a basic physiologically based pharmacokinetic (PBPK) model (J. C. Ramsey and M. E. Andersen, 1984, Toxicol. Appl. Pharmacol. 73, 159-175) to characterize the biological determinants of D4 kinetics. Parameter estimation techniques indicated an unusual set of characteristics, i.e., a low blood:air (P(b:a) congruent with 0.9) and a high fat:blood partition coefficient (P(f:b) congruent with 550). These parameters were then determined experimentally by equilibrating tissue or liquid samples with saturated atmospheres of D4. Consistent with the estimates from the time-course data, blood:air partition coefficients were small, ranging from 1.9 to 6.9 in six samples. Perirenal fat:air partition coefficients were large, from 1400 to 2500. The average P(f:b) was determined to be 485. This combination of partitioning characteristics leads to rapid exhalation of free D4 at the cessation of the inhalation exposure followed by a much slower redistribution of D4 from fat and tissue storage compartments. The basic PK model failed to describe D4 tissue kinetics in the postexposure period and had to be expanded by adding deep-tissue compartments in liver and lung, a mobile chylomicron-like lipid transport pool in blood, and a second fat compartment. Model parameters for the refined model were optimized using single-exposure data in male and female rats exposed at three concentrations: 7, 70, and 700 ppm. With inclusion of induction of D4 metabolism at 700 ppm (3-fold in males, 1-fold in females), the parameter set from the single exposures successfully predicted PK results from 14-day multiple exposures at 7 and 700 ppm. A common parameter set worked for both genders. Despite its very high lipophilicity, D4 does not show prolonged retention because of high hepatic and exhalation clearance. The high lipid solubility, low blood:air partition coefficient, and plasma lipid storage with D4 led to novel distributional characteristics not previously noted for inhaled organic hydrocarbons. These novel characteristics were only made apparent by analysis of the time-course data with PBPK modeling techniques.

Disposition of radioactivity in fischer 344 rats after single and multiple inhalation exposure to [(14)C]Octamethylcyclotetrasiloxane ([(14)C]D(4)).

The retention, distribution, metabolism, and excretion of [(14)C]octamethylcyclotetrasiloxane (D(4)) were studied in Fischer 344 rats after single and multiple exposures to 7, 70, or 700 ppm [(14)C]D(4). Subset groups were established for body burden, distribution, and elimination. Retention of inhaled D(4) was relatively low (5-6% of inhaled D(4)). Radioactivity derived from [(14)C]D(4) 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 dose-dependent shifts in elimination pathways were seen. After multiple exposures, the elimination pathways were dose- and gender-independent. These data define possible pathways for metabolism of D(4) and allow estimation of the persistence of D(4) and/or its metabolites in rats.

Identification of metabolites of octamethylcyclotetrasiloxane (D(4)) in rat urine.

Octamethylcyclotetrasiloxane (D(4)) is an industrial chemical of significant commercial importance. In this study, its major urinary metabolites were identified. The urine samples described here were collected from male and female Fischer rats (F-344) administered [(14)C]D(4) i.v. The metabolite profile was obtained using an HPLC system equipped with a radioisotope detector. HPLC analysis was performed on a C18 column, using an acetonitrile/water mobile phase. The HPLC radiochromatogram revealed two major and at least five minor metabolites. The two major metabolites, constituting 75 to 85% of the total radioactivity, were identified as dimethylsilanediol [Me(2)Si(OH)(2)] and methylsilanetriol [MeSi(OH)(3)]. Formation of MeSi(OH)(3) clearly established demethylation at the silicon-methyl bonds of D(4). No parent D(4) was present in urine. The minor metabolites identified were tetramethyldisiloxane-1,3-diol [Me(2)Si(OH)-O-Si(OH)Me(2)], hexamethyltrisiloxane-1,5-diol [Me(2)Si(OH)-OSiMe(2)-OSi(OH)Me(2)], trimethyldisiloxane-1,3,3-triol [MeSi(OH)(2)-O-Si(OH)Me(2)], dimethyldisiloxane-1,1,3,3-tetrol [MeSi(OH)(2)-O-Si(OH)(2)Me], and dimethyldisiloxane-1,1,1,3,3-pentol [Si(OH)(3)-O-Si(OH)(2)Me]. The structural assignments were based on gas chromatography-mass spectrometry analysis of the tetrahydrofuran metabolite extracts, which were derivatized using bis(trimethylsiloxy)triflouroacetamide, a trimethylsilylating agent. The structures were confirmed by synthesizing (14)C-labeled standards and comparing their HPLC radiochromatograms with the corresponding components in the rat urine. GC-MS spectral comparisons of the trimethylsilylated derivatized standards and urinary components also were made to further confirm their identities. Finally, several of the urinary metabolites were fractionated using HPLC, and GC-MS comparisons were again made for positive structural identification. The pathways for metabolite formation are not yet understood, but a mechanistic hypothesis has been proposed to account for the various metabolites observed thus far.

Extraction of octamethylcyclotetrasiloxane and its metabolites from biological matrices.

Octamethylcyclotetrasiloxane (D4) is an industrial chemical of significant commercial importance. It is a key ingredient in a variety of product formulations and a critical intermediate in the production of silicone polymers. As part of the pharmacokinetic investigation of its disposition and metabolism, an efficient extraction methodology has been developed to recover both the parent D4 and its metabolites from various biological matrices, including blood, plasma, urine, feces, liver, lung, and fat. Of the common organic solvents evaluated (hexane, methylene chloride, diethylether, chloroform, methylisobutylketone, etc.), tetrahydrofuran (THF) was demonstrated to be an excellent choice for extracting both D4 and its metabolites. Test samples were either spiked with 14C-D4 or obtained from animals administered 14C-D4 via various exposure routes (inhalation, i.v., etc.) and then subjected to THF extraction. Recoveries of D4 and its unbound metabolites from plasma were essentially quantitative in three extractions. Even in a single extraction, the recoveries were > 90%. Similar results were obtained for other matrices as well. Extraction efficiencies were measured as follows: lung, 98.2 +/- 0.3%; liver, 95.4 +/- 0.4%; fat, 99.4 +/- 0.8%; urine, 98.1 +/- 0.2%; feces, 94.1 +/- 0.6%. HPLC profiles obtained before and after extraction were essentially identical, further confirming the suitability of THF as an extractant.

Biodistribution, metabolism, and excretion of radioiodinated phospholipid ether analogs in tumor-bearing rats.

The phospholipid ether analog, [125I]-1-O-[12-(m-iodophenyl)dodecyl]propanediol-3-phosphocholine (NM-295) was synthesized and evaluated for its ability to visualize tumors. Preliminary studies were performed in rats bearing the Walker 256 carcinosarcoma. Most of the radioactivity was cleared from the animals during the first 24 hours. However, the tumor showed a decreased rate of clearance of radioactivity when compared with non-target tissue. This difference in the clearance rate allowed for excellent images of the tumor at 24 hours. Scintigraphic images compared favorably with other radioiodinated phospholipid ether analogs such as [125I-rac-1-O-[12-(m-iodophenyl)dodecyl]-2-O-methylglycero-3- phosphocholine (NM-294) and [125I]-12-(m-iodophenyl)dodecyl phosphocholine (NM-324). In contrast with the latter two compounds, however, tissue distribution studies revealed that NM-295 cleared at a much faster rate from all tissues, including tumor. In addition, within 24 hours following administration of NM-295, over 70% of the radioactivity was excreted as compared to 50% and 20% for NM-294 and NM-324, respectively. The majority of excreted radioactivity appeared in the urine for all three compounds. Thin-layer chromatography of urine and fecal extracts showed the presence of metabolites only. In contrast, lipid extracts of either liver or tumor demonstrated only the presence of the parent compound. Therefore, these data suggest that in each case it was the parent phospholipid analog that was taken up and retained by the tissues, while the metabolic product(s) was cleared and excreted from the animal.

Selective localization of a radioiodinated phospholipid ether analog in human tumor xenografts.

Administration of [125I]-rac-1-O-[12-(m-iodophenyl)dodecyl-2-O-methylglycero-3- phosphocholine (NM-294) to athymic mice implanted with human tumors of several histologies, including adenocarcinoma of the ovary and colon, melanoma and small-cell carcinoma of the lung, resulted in excellent images of the tumors by gamma camera scintigraphy. Images of the tumor were obtained at 5 days or more postinjection, by which time nearly all background activity had cleared from the liver and gastrointestinal tract. Tumor-to-blood ratios at this time were quite high and ranged from approximately 8:1 (melanoma) to 30:1 (ovarian carcinoma), which is consistent with the scintigraphic images obtained in all human tumor models. Lipid extraction of the liver and tumor at 13 days postinjection showed that most of the radioactivity in these tissues remained associated with the parent compound, with only a small amount retained by the liver. Appropriately radioiodinated NM-294 has substantial potential as a tumor-avid radiopharmaceutical.

Selective localization of radioiodinated alkylphosphocholine derivatives in tumors.

We have designed and synthesized two radioiodinated analogs of hexadecylphosphocholine in order to evaluate their tumor imaging potential. 12-(m[125I]iodophenyl)dodecyl phosphocholine (NM-324) and hexadecyl-2-[N,N-dimethyl-N-(m[125I]iodobenzyl)-ammonium] ethyl phosphate (NM-326) demonstrated the ability of such compounds to localize in and thereby visualize the Walker 256 tumor in rats. However, the tumor avidity of NM-324 was far superior to NM-326. In addition, NM-324 showed excellent tumor localization in athymic mice bearing subcutaneous human tumors.