Introduction

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Hexamethyldisiloxane (MM, HMDS¹), the smallest member of the polydimethylsiloxane polymers, and decamethylcyclopentasiloxane (D₅), a cyclic siloxane are colorless volatile fluids. MM is quite volatile with a vapor pressure of 42.2 mm Hg at 25°C and a boiling point of 100°C (Flanningam, 1986). D₅ is relatively less volatile with a vapor pressure of 2 mm Hg at 50°C and a boiling point of 210°C. The aqueous solubilities of MM and D₅ are 930 and 17 ppb, respectively (Varaprath et al., 1996). The primary use of MM and D₅ is as intermediates in the manufacturing of high molecular weight siloxane polymers. MM and D₅ also find use as vehicles or ingredients in a wide range of consumer product formulations (Cameron et al., 1986) since they have several favorable properties such as low surface tension, adequate evaporation rate, lack of odor, high degree of compatibility with many consumer product ingredients, and low toxicity. Typical examples of applications include moisturizing creams, lotions, bath oils, colognes, shaving products, and perfumes. Besides these product applications, they are also used as cleaners, lubricants, and penetrating oils.

The rigorously purified MM (Dow Corning OS-10, purity >99.9%) is one of the many ozone-safe volatile methylsiloxanes that is exempt from federal volatile organic compound regulations and hence is accepted as an alternative for other organic solvents. Another important industrial use of MM is as a chain-terminating agent in siloxane polymerizations. The use of MM and D₅ in various product formulations necessitated conducting chemical and environmental fate/effects tests of them.

Potential human exposure to MM and D₅ can result at the work place during the manufacturing process, as well as through the normal use of consumer products that contain them. Only sparse toxicological information is available on these siloxanes since they are believed to be relatively inert and of low toxicity. However, octamethylcyclotetrasiloxane (D₄), a homolog of D₅ had been extensively studied. In rodents, inhalation exposure to D₄ results in dose-related hepatomegaly, transient hepatic hyperplasia, hypertrophy, and induction of hepatic cytochrome P450 enzymes in a fashion similar to phenobarbital (McKim et al., 1998, 2001). Very limited toxicity data are available on HMDS and D₅ in biological systems. In a 13-week subchronic MM whole-body inhalation, renal histopathology consistent with male rat-specific -2U-globulin nephropathy accompanied by slight increases in plasma urea and creatinine concentrations were seen in male Fischer F344 rats at vapor concentrations of 600 to 5000 ppm. No other treatment-related pathological changes were seen in MM-exposed rats (Cassidy et al., 2001).

In a 13-week subchronic D₅ inhalation study (Burns-Naas et al., 1998) with male and female Fischer F344 rats, exposure-related increases in absolute and/or relative liver weights were observed in both sexes, although histopathology of the liver was uneventful. The histopathology evaluation following D₅ inhalation exposure indicated lung as the primary target organ. An increase in focal macrophage accumulation and interstitial inflammation were observed in the lungs of male and female rates at high concentrations (224 ppm) of D₅.

A comprehensive program has been initiated to assess the kinetics, metabolism and toxicity of MM and D₅ in rats after relevant routes of exposure. To aid in the pharmacokinetic investigations, identification of MM and D₅ metabolites in urine collected from rats following exposure to these materials were undertaken, and the results are presented in this paper.

	Materials and Methods

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Instrumentation/Reagents. Radioactivity measurements were made using a liquid scintillation counter (Packard, model 2500 TR; PerkinElmer Life Sciences, Boston, MA). HPLC analyses were performed with a Hewlett Packard 1050 liquid chromatograph (Hewlett Packard, Palo Alto, CA) equipped with an HP autosampler (model 79855A), and a Radiomatic (model 500 TR series) flow scintillation analyzer from PerkinElmer Life Sciences. The detector was installed with a flow liquid cell 500 µl in size. HPLC conditions in the water/acetonitrile mobile phase were as follows: 100% water, 0 to 20 min; 100% water to 100% acetonitrile, 20 to 40 min; 100% acetonitrile, 40 to 50 min; 100% acetonitrile to 100% water, 50 to 60 min. A C₁₈ Alltima column (4.6 × 250 cm and 5 µm from Alltech Associates, Deerfield, IL) was used as the stationary phase; Ultima-Flo M liquid scintillation cocktail was used in the flow cell. The ratio of column effluent to scintillation cocktail was 3:1.

Dose Administration and Sample Collection. Urine samples used in the metabolite investigation were from female rats administered [¹⁴C]MM by two routes, oral gavage and i.v.

Solvent Extraction of MM Metabolites. A 500-mg aliquot was measured into a 7-ml glass vial with an aluminum-lined screw cap. Dichloromethane (2 ml) was added. The vial was then tightly capped, and the contents were first vortex mixed at high-speed settings for 3 min and then centrifuged (3000 RPM) for 4 min. The clear bottom layer of CH₂Cl₂ was carefully removed with a Pasteur pipette and collected in a clean 20-ml vial. Fresh CH₂Cl₂ was added to the sample residue, and the extraction procedure was repeated as before. Extractions were repeated until no significant amount of radioactivity was left in the urine residues. The extracts were combined, treated with anhydrous MgSO₄ to remove water, vortex mixed (4-5 min), and finally centrifuged (5 min) to obtain a clear, dry CH₂Cl₂ extract of the metabolites. The dry extract was then concentrated to about 250 to 300 µl by gently blowing a nitrogen stream via Pasteur pipette along the sides of the container vial. Extraction was also performed on aliquots of samples in a similar fashion using THF.

Solid Phase Extraction of D₅ Metabolites. Since most of the D₅ metabolites are not soluble in CH₂Cl₂, the extractions were performed with THF only. Also, in an effort to increase the efficiency, solid phase extractions (Varaprath and Cao, 2000) were performed as follows.

Derivatization. The dried and concentrated CH₂Cl₂ or THF extract of the metabolites was treated with an equivalent amount (v/v) of BSTFA, vortex mixed for 2 to 5 min, and then shaken using a horizontal shaker for 2 h at ambient temperatures. BSTFA treatment was repeated as needed, if partial derivatization became apparent from GC-MS analyses. Derivatization with specially purified hexamethyldisiloxane (99.9%) was carried out in a similar way, except that a catalytic amount of 10% (w/w) hydrochloric acid or CF₃SO₃H was also added. The reagent hexamethyldisiloxane was used only to derivatize a metabolite such as dimethylsilanediol containing one Si per molecule, since other metabolites containing Si-O-Si bonds will undergo chemical transformation (hydrolysis) under acidic conditions. The trimethylsilyl derivatives of the metabolite sample were then subjected to analysis by GC-MS for structural identification.

Synthesis of Metabolite Standards and Precursors. Synthesis of the standards for identification of metabolites of MM are described below with the exception of [¹⁴C]Me₂Si(OH)₂ that was synthesized following the literature procedure (Varaprath, 1999). It should be pointed out unless otherwise indicated, no special attempts were made to isolate or purify metabolite standards and precursors from the by-products. Their presence was verified from GC-MS and in some cases by HPLC radiochromatographic analyses. All the metabolites of D₅ with the exception of nonamethylcyclopentasiloxanol and hydroxymethylnonamethylcyclopentasiloxane were synthesized following the literature procedure (Varaprath et al., 1999). Nonamethylcyclopentasiloxanol was available internally at Dow Corning Corp. (Midland, MI). Hydroxymethylnonamethylcyclopentasiloxane was inferred by GC-MS analysis.

Unlabeled HOCH₂SiMe₂OSiMe₂CH₂OH (1,3-bis(Hydroxymethyl)tetramethyldisiloxane). The chloro precursor ClCH₂SiMe₂OSiMe₂CH₂Cl (23.1 g) was placed in a one-necked 250-ml round bottomed flask equipped with a water condenser, a magnetic stir bar, and a Drierite moisture trap. Potassium acetate (19.6 g), glacial acetic acid (50 ml), and a catalytic amount (0.5 g) of potassium iodide were added. The contents were heated to a gentle reflux for 48 h using an oil bath, and then the flask was cooled to room temperature. The solution was transferred to a 500-ml separatory funnel. The contents were washed with water (3 × 50 ml). Pentane (50 ml) was added, and the solution was washed with a 5% sodium bicarbonate solution. The organic layer was then washed several times with water until the wash water remained neutral to a pH paper. Pentane was removed using a rotary evaporator at 40°C. Pure product (23 g, 80%) AcOCH₂SiMe₂OSiMe₂CH₂OAc was obtained by distillation at 85°C/1.6 mm Hg.

[¹⁴C]HOCH₂SiMe₂OH (Hydroxymethyldimethylsilanol). Urine containing MM metabolites was subjected to fractionation by HPLC. The fraction eluting during the time interval of 25.5 to 28.0 min that contained [¹⁴C]HOCH₂SiMe₂-O-SiMe₂CH₂OH was collected from repeat injections. The individual fractions were combined. A 500-µl aliquot of the combined fraction was placed in a 7-ml vial with Teflon-lined screw cap. A 10% HCl (20 µl) was added, and the contents were shaken for 2 h in a water bath maintained at 50°C to generate the [¹⁴C] HOCH₂SiMe₂OH in aqueous solution. Unlabeled HOCH₂SiMe₂OH was synthesized by hydrolysis at 37°C for an hour of HOCH₂SiMe₂OSiMe₂CH₂OH (50 µl) with rat liver microsomes (50 µl) and NADPH (2 mM) in a pH 7.7 phosphate buffer (800 µl) containing MgCl₂. Product was extracted with 1 ml of THF in presence of NaCl (2 g). GC-MS analysis showed the formation of desired product.

[¹⁴C]Me₃SiOSiMe₂CH₂OH (Hydroxymethylpentamethyldisiloxane). MM (300 µl) was added to the aqueous solution of [¹⁴C]-HOCH₂SiMe₂OH synthesized above, and the contents were shaken at ambient temperature for 18 h using the Eberbach horizontal shaker. The sample was centrifuged for 4 min, and the MM layer was collected and treated with anhydrous Na₂CO₃. HPLC analysis of the MM solution showed that the product [¹⁴C]Me₃SiOSiMe₂CH₂OH eluted at 39.1 min. GC-MS analysis confirmed its presence. The same product was also obtained shaking (3.5 h) HOCH₂SiMe₂OSiMe₂CH₂OH (50 µl) and [¹⁴C]Me₃SiOSiMe₃ (200 µl) in the presence of CF₃SO₃H. The product was extracted with ether and neutralized with anhydrous Na₂CO₃.

Unlabeled Me₃SiOSiMe₂CH₂OH. Diethyl ether (250 ml) and potassium trimethylsilanolate (32 g) were placed in a 500-ml round bottomed flask equipped with an addition funnel and a magnetic stir bar. Chloromethyldimethylchlorosilane (35.5 g) was added dropwise. The reaction mixture was stirred overnight at room temperature. It was filtered to remove the solid by-product. The filtrate was collected and washed with water until the wash water was neutral. The filtrate was distilled to obtain chloromethylpenatmethyldisiloxane (20 g). The latter (18.07 g) was converted to Me₃SiOSiMe₂CH₂OCOCH₃ (following the procedure described above for HOCH₂SiMe₂OSiMe₂CH₂OH) using potassium acetate (9.22 g), potassium iodide (200 mg), and glacial acetic acid (20 ml). The ether extract dried over anhydrous MgSO₄ was distilled (42°C/4 mm) to obtain the pure acetate. Using 5.6 g of the acetoxy derivative thus prepared, 1.1 g of NaBH₄ in 5 ml THF and 5.15 g of BF₃-THF (a 3M solution in THF from Aldrich Chemical Co.), the procedure described above for HOCH₂SiMe₂OSiMe₂CH₂OH was followed to obtain the desired Me₃SiOSiMe₂CH₂OH.

[¹⁴C]Me₃SiOH (Trimethylsilanol). A 250-µl solution of [¹⁴C]Me₃SiOSiMe₃ in acetonitrile was placed in a 2-ml Nalgene vial with a screw cap. A 10% (w/w) HCl solution (500 µl) was added. The contents were shaken overnight for 14 h. A 200-µl aliquot of the acidic solution of [¹⁴C]Me₃SiOH was then neutralized with anhydrous Na₂CO₃. The product eluted at 31.90 min by HPLC.

[¹⁴C]Me₃SiOSiMe₂OH (Pentamethyldisiloxanol). 1,1,3,3-Tetramethyldisiloxane (166 mg), [¹⁴C]Me₃SiOSiMe₃ (405 mg solution in unlabeled MM), and 10% HCl (10 ml) were placed in a 20-ml scintillation vial and shaken at room temperature for 24 h using a horizontal shaker. The aqueous phase was discarded. The organic phase was washed with water until wash water was neutral. The reaction mixture was placed in a 10 ml beaker and while stirring, a slurry of 92 mg of 10% Pd on charcoal in 1 ml of water was added. It was filtered, and the filtrate was subjected to fractionation by HPLC. The fraction eluting at retention time 38.7 min was collected from repeat injections. The individual fractions were combined.

Unlabeled Me₃SiOSiMe₂OH. Water (803 ml) was placed in a 3-liter round-bottomed flask equipped with a magnetic stir bar and an addition funnel. The flask was cooled to 0°C. Water was vigorously stirred, and a mixture of Me₃SiCl (120 g, 0.904 mol) and Me₂SiClH (209 g) was added slowly via the addition funnel. The temperature of the reaction mixture was maintained below 10°C. Following the addition, the stirring was continued for 4 h, and the contents were allowed to warm to ambient temperature. The organic layer was separated, washed with water until the wash water was neutral to a pH paper. The solution was dried with anhydrous Na₂SO₄ overnight and distilled using a spinning band column to obtain the intermediate Me₃SiOSiMe₂H. A slurry of 10% palladium on carbon (0.35 g) in 3 ml of dioxane was placed in a 500-ml beaker and stirred using a magnetic stir bar. The silane Me₃SiOSiMe₂H (50 g) was added slowly, and the temperature of the reaction mixture was maintained at room temperature by cooling the beaker in a water bath. After the brisk effervescence ceased, the beaker was covered with aluminum foil and the stirring continued overnight. The mixture was stirred with anhydrous Na₂SO₄ (2 h) and filtered through a bed of anhydrous MgSO₄. The filtrate was subjected to distillation. Pentane and dioxane were removed at 25°C/60 mm Hg and 30°C/32 mm Hg, respectively. The product Me₃SiOSiMe₂OH was distilled at 54°C/16 mm Hg.

	Results

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Metabolites of MM.

HPLC profile of MM metabolite Using a radioisotope detector, HPLC analyses of the urine samples (from oral as well as i.v.-dosed rats) containing metabolites of MM revealed the presence of several metabolites. For the purpose of illustration, the profile obtained from one of the rats administered orally with [¹⁴C]MM is shown in Fig. 1. There were some minor variations with respect to the presence of trace levels of metabolites in the HPLC profiles among rats administered MM by the same route as well as between those administered MM via different routes. In spite of the variations, there were 5 metabolites that were commonly present in all samples. These were the metabolites eluting at retention times centered around 4.0, 13.10, 24.2, 31.9, and 39.0 min. These metabolites constituted 72 to 74% and 80 to 85% of the total radioactivity eluted in HPLC for the oral and intravenous routes, respectively. The major focus of this work was to establish the structure of these common metabolites. Other metabolites revealed from GC-MS analyses, the retention times of which were not assigned in HPLC, are also presented. The HPLC and GC-MS retention times of the metabolites are compiled in Table 1. The total radioactivity of the metabolites eluted in HPLC from a known volume of urine sample accounted for essentially 100% of the activity measured for the same volume of urine by liquid scintillation.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   HPLC radiochromatogram of the metabolites of hexamethyldisiloxane in Fischer 344 female rat urine. A, HOCH2SiMe2OH; B, HOSiMe2OH; C, HOCH2SiMe2OSiMe2CH2OH; F, Me3SiOH; I, Me3SiOSiMe2CH2OH; peaks D, E, G, and H were not assigned due to unavailability of radiolabeled standards.

Identification of MM metabolites². For structure elucidation by GC-MS, the metabolites were first extracted into an organic solvent. Extraction was performed using THF as well as methylene chloride. Although the utility of THF as an efficient solvent for extraction of urinary metabolites of silicones has already been established (Varaprath et al., 1998), we have experimentally determined that THF was also quite efficient in extracting metabolites of MM from urine. The extraction efficiency for MM metabolites from urine was determined to be 97.7 ± 0.3%. However, since a preliminary investigation revealed the presence of several metabolites containing hydroxymethyl functions (CH₂OH), which are readily soluble in dichloromethane, the latter was also used on fresh aliquots of samples for extraction. Use of methylene chloride significantly reduced the presence of endogenous materials in the extracts. Unlike the Si-OH functional metabolites, the CH₂OH functional metabolites readily elute by GC. Therefore the extracts were analyzed as such by GC-MS, in addition to analyzing them following trimethylsilyl derivatization. The details on the structural elucidation aspects for the individual metabolite identified are given below.

Metabolite HOCH₂SiMe₂OSiMe₂CH₂OH (1,3-bis(hydroxymethyl) tetramethyldisiloxane). GC-MS profile of a methylene chloride extract of the urine sample revealed a component of m/z 163 (M-CH₂OH) at a retention time of 7.6 min. It was established from HPLC fractionation of the urine sample, followed by methylene chloride extraction and GC-MS analysis, that this component in urine eluted at 24.6 min. The GC-MS retention time and spectral characteristic of this component matched with the synthetic material (Fig. 2). The trimethylsilyl derivatives of the synthetic standard and of the 24.6-min metabolite fraction also matched with respect to retention time and mass fragmentation patterns. The trimethylsilyl derivatization gave rise to mass m/z 323 (M-CH₃) expected for the fully derivatized molecule of the structure Me₃SiOCH₂SiMe₂OSiMe₂CH₂OSiMe₃ (mol. wt. = 338).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Mass spectrum of the urinary component eluting at 26.5 min in HPLC identified as HOCH2SiMe2OSiMe2CH2OH. m/z fragments are as follows: 179, M-CH3; 163, M-CH2OH; 89, M-OSiMe2CH2OH.

Metabolite HOSiMe₂CH₂OH (hydroxymethyldimethylsilanol). The component eluting in HPLC at retention time 4.0 min in Fig. 1 was assigned the structure HOSiMe₂CH₂OH. Oxidation of one of the methyl groups in MM followed by hydrolysis of Si-O-Si linkage can give rise to HOSiMe₂CH₂OH. Its presence was confirmed by its synthesis by two different routes.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Mass spectrum of the urinary component eluting at 4 min in HPLC (identified as HOMe2SiCH2OH) that was protected with a trimethylsilyl group to obtainMe3SiOSiMe2CH2OH. m/z fragments are as follows: 163, M-CH3; 147, M-CH2OH; 73, Me3Si.

Metabolite HOSiMe₂OH (dimethylsilanediol). An authentic sample of [¹⁴C]HOSiMe₂OH was available and its HPLC retention time (13.1 min) matched with one of the metabolite components in urine sample. When the urine sample was fortified with this standard and analyzed by HPLC, coelution at 13.1 min was observed. For structural analysis, the component in urine eluting at 13.1 min was collected by HPLC fractionation and subjected to trimethylsilyl derivatization with MM in presence of a catalytic amount of CF₃SO₃H. The organic layer containing the derivative was analyzed by GC-MS following neutralization with anhydrous Na₂CO₃ and it showed the expected formation of Me₃SiOSiMe₂OSiMe₃ (m/z 221 from M-CH₃). The retention time and mass spectral characteristics of the latter matched to a commercially available standard thus confirming the presence of the metabolite HOSiMe₂OH in urine.

Metabolite Me₃SiOH (trimethylsilanol). The component in urine eluting at 31.9 min in HPLC was unambiguously assigned the structure as [¹⁴C]Me₃SiOH by comparison with a synthetic standard. An acetonitrile solution of [¹⁴C]Me₃SiOSiMe₃ was hydrolyzed using a 10% HCl solution and then neutralized with solid sodium carbonate to obtain [¹⁴C]Me₃SiOH as an aqueous solution. This synthetic material eluted at 31.9 min and matched to the component in urine. The metabolite Me₃SiOH is highly volatile, and due to its loss during concentration, the extracts of urine containing MM metabolites did not reveal its presence in GC-MS analysis.

Metabolite Me₃SiOSiMe₂OH (pentamethyldisiloxanol). Analysis of the methylene chloride extract of urine by GC-MS showed a component eluting at 3.17 min. This was assigned the structure Me₃SiOSiMe₂OH based on the mass fragments 149 (M-CH₃) and 133 (M-CH₃O). Comparison of its GC-MS data (Fig. 4) with an authentic sample synthesized confirmed the structure of this metabolite in urine.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Mass spectrum of the component identified as Me3SiOSiMe2OH in CH2Cl2 extract of urine. m/z fragments are as follows: 149, M-CH3; 133, M-OCH3; 75, M-OSiMe3.

Metabolite Me₃SiOSiMe₂CH₂OH (hydroxymethylpentamethyldisiloxane). The component eluting at 4.84 min in GC-MS analysis of the methylene chloride extract of urine was assigned the structure Me₃SiOSiMe₂CH₂OH based on the mass fragments 163 (M-CH₃) and 147 (M-CH₂OH) expected for the siloxane moiety of mass 178. The GC-MS data were identical to that in Fig. 3 and also matched with that of an authentic sample synthesized thus confirming the structure of this metabolite present in urine.

Other Metabolites of MM Revealed by GC-MS Analysis. There were a few minor metabolites for which HPLC data comparisons could not be made due to lack of availability of ¹⁴C-labeled standards. However, their presence was apparent from the GC-MS analysis. The presence of these metabolites was confirmed by comparison of the GC-MS data with the synthetic materials that were not labeled.

Metabolite HOSiMe₂OSiMe₂CH₂OH (3-hydroxymethyl-1,1,3,3 tetramethyldisiloxanol). From the list of the confirmed metabolites that indicated the presence of CH₂OH and OH functions, it was logical to expect a metabolite of the structure HOSiMe₂OSiMe₂CH₂OH to be formed from MM with Si-O-Si linkage intact. THF extract of urine did show by GC-MS a component eluting at 6.41 min with mass fragments 165 (M-CH₃) and 149 (M-CH₂OH) expected for this siloxane moiety of mol. wt. 180. The synthetic material eluted at 6.37 min, and its fragmentation pattern was identical to the 6.41-min component in urine (Fig. 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Mass spectrum of the component identified as HOMe2SiOSiMe2CH2OH in CH2Cl2 extract of urine. m/z fragments are as follows: 165, M-CH3; 149, M-CH2OH; 75, M-OSiMe2CH2OH.

Metabolite 2,2,5,5-tetramethyl-2,5-disila-1,3-dioxalane. GC-MS data on the methylene chloride extract of urine showed a component eluting at 3.52 min of m/z 162. This component was assigned the structure shown below.

View larger version (9K): [in this window] [in a new window]   Scheme 1.   2,2,5,5-Tetramethyl-2,5-disila-1,3-dioxalane.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Mass spectrum of the component identified as 2,2,5,5-tetramethyl-2,5-disila1,3-dioxalane in CH2Cl2 extract of urine. m/z fragments are as follows: 162, M-CH3; 73, Me3Si.

Metabolite 2,2,5,5-tetramethyl-1,4-dioxa-2,5-disilacyclohexane. GC-MS data on the THF extract of urine showed a component eluting at 5.78 min with a mass of 176. This component was assigned the structure shown below:

View larger version (9K): [in this window] [in a new window]   Scheme 2.   2,2,5,5-Tetramethyl-1,4-dioxa-2,5-disilacyclohexane.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Mass spectrum of the component identified as 2,2,5,5-tetramethyl-1,4-dioxa-2,5-disilacyclohexane. m/z fragments are as follows: 176, M; 161, M-CH3.

Metabolites of D₅.

HPLC profile of D₅ metabolites The HPLC radiochromatogram for D₅ metabolites is shown in Fig. 8. It revealed two major metabolites (A and C) and at least three minor metabolites (B, D, and E). Combined, the two major components A and C constituted ~75% of the total radioactivity and B, D, and E accounted for the rest. The total radioactivity of the metabolites eluted in HPLC from a known volume of urine sample accounted for essentially 100% of the activity measured for the same volume of urine by liquid scintillation. It should be pointed out that although HPLC showed essentially five metabolites, there were other metabolites that were revealed by GC-MS but present at levels below detection by HPLC.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   HPLC radiochromatogram of the metabolites of decamethylcyclopentasiloxane in Fischer 344 female rat urine. A, MeSi(OH)3; B, Me(OH)2SiOSi(OH)3; C, Me2Si(OH)2; Me2(OH)Si-SiMe(OH)2; HO(SiMe2O)3-H.

Identification of D₅ metabolites³. As far as the major metabolites were concerned, the HPLC profile of D₅ metabolites were essentially identical to that reported by the author for D₄ in rat urine (Varaprath et al., 1999). The HPLC components of identical retention times were therefore expected to have the corresponding structures. The methodology used in D₄ metabolites identification was applied to the case of D₅. The hydroxy groups of the metabolites were protected with trimethylsilyl groups, and structures of the resultant derivatives were assigned by comparison to that of the standards. The GC-MS retention times of the TMS derivatives of all the metabolites and their respective major mass (m/z) fragments are compiled in Table 2.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Mass spectrum of the trimethylsilyl-protected nonamethylcyclopentasiloxanol. m/z fragments are as follows: 429, M-CH3; 73, Me3Si.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Mass spectrum of the trimethylsilyl-protected hydroxymethylnonamethyl-cyclopentasiloxane. m/z fragments are as follows: 443, M-CH3; 355, M-CH2OSiMe3.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Since potential human exposure to MM and D₅ can result at the work place during the manufacturing process, as well as through the normal use of consumer products that contain them, comprehensive pharmacokinetic studies combined with toxicity studies will be helpful in characterizing the risk, if any, to human populations exposed to MM and D₅. Preliminary pharmacokinetic data following a single inhalation exposure of 5000 ppm [¹⁴C]MM to male and female Fischer F344 rats (unpublished data) has indicated that most of the recovered radioactivity was in urine (~40%) and expired volatiles (~50%), with minor amounts, either excreted via CO₂ (1-2%) and feces (1-2%) or remaining in the carcass (~5%) 168-h post exposure.

In the case of D₅, following a single inhalation exposure of 160 ppm [¹⁴C]D₅ to male and female Fischer F344 rats, most of the recovered radioactivity (unpublished data) was found in urine (~30%) or excreted in feces (~50%). The remaining radioactivity was either excreted via expired volatiles (4-10%), CO₂ (5%), or remained in the carcass (10%) after 168-h post exposure. The disposition of radioactivity for both MM and D₅ following inhalation exposure is similar to that observed with other siloxanes (Bennett and Aberg, 1975; Plotzke et al., 2000; Andersen et al., 2001).

More comprehensive mass balance studies and pharmacokinetics analysis on MM and D₅ from various routes of administration are currently underway and will be published later. The objective of the work presented in this paper is to identify the major metabolites of MM and D₅ in urine collected from rats following exposure, since metabolism plays a significant role in elimination of these materials from the body.

As demonstrated by the HPLC profile, with both MM and D₅ the radioactivity that was excreted in the urine contained only polar metabolites and no parent material. It was apparent from the results that except for the commonly present dimethylsilanediol, the urinary metabolites of the linear siloxane MM were different from those obtained for cyclic siloxane D₅. Presence of a hydroxymethyl group, the primary oxidation product of the methyl group, was found in most of the metabolites of MM. The bis(hydroxymethyl) metabolite with Si-O-Si bond in tact was the major metabolite. Some metabolites, in addition, have hydroxy groups. With D₅ on the other hand, presence of multiples of hydroxy groups was a common feature. Metabolites containing CH₂OH groups were absent with the exception of the presence of D₄D'CH₂OH at trace level. The reasons for the meager presence of CH₂OH functional metabolites in D₅ are not clear, but it may be related to factors such as relative stability when the CH₂OH is on a ring system and solubility of the metabolites. In a smaller cyclic siloxane D₄ (Varaprath et al., 1999), the corresponding metabolite D₃D'CH₂OH was not detected.

The possible pathways for the formation of the metabolites of MM and D₅ are shown in Figs. 11 and 12. Metabolites such as 2,2,5,5-tetramethyl-2,5-disila-1,3-dioxalane and 2,2,5,5-tetramethyl-1,4-dioxa-2,5-disilacyclohexane were unexpected. Due to lack of availability of ¹⁴C-labled standards, it was not established that these metabolites were actually present in urine. It is quite possible that the mixed ether linkage (CH₂-O-Si) make these metabolites quite water soluble and thus contribute to their formation and subsequent elimination in urine. As shown in the mechanistic scheme, these metabolites could well be artifacts arising from inadvertent cyclization, at the injection port of the GC-MS, of the corresponding linear metabolites containing OH and CH₂OH present in urine. The presence of metabolites such as dimethylsilanediol in MM and methylsilanetriol in D₅ clearly established that some demethylation occurs at the silicon-methyl bond.

View larger version (21K): [in this window] [in a new window]   Fig. 11.   Possible pathways for the formation of the metabolites of hexamethyldisiloxane in Fischer 344 rat urine.

View larger version (13K): [in this window] [in a new window]   Fig. 12.   Possible pathways for the formation of the metabolites of decamethylcyclopentasiloxane in Fischer 344 rat urine.