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Vol. 27, Issue 11, 1267-1273, November 1999
Dow Corning Corporation (S.V., K.P.P., S.N.), Midland, Michigan; and SmithKline Beecham (K.L.S.), King of Prussia, Pennsylvania
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Octamethylcyclotetrasiloxane (D4) 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 [14C]D4 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 [Me2Si(OH)2] and methylsilanetriol [MeSi(OH)3]. Formation of MeSi(OH)3 clearly established demethylation at the silicon-methyl bonds of D4. No parent D4 was present in urine. The minor metabolites identified were tetramethyldisiloxane-1,3-diol [Me2Si(OH)-O-Si(OH)Me2], hexamethyltrisiloxane-1,5-diol [Me2Si(OH)-OSiMe2-OSi(OH)Me2], trimethyldisiloxane-1,3,3-triol [MeSi(OH)2-O-Si(OH)Me2], dimethyldisiloxane-1,1,3,3-tetrol [MeSi(OH)2-O-Si(OH)2Me], and dimethyldisiloxane-1,1,1,3,3-pentol [Si(OH)3-O-Si(OH)2Me]. 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 14C-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.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Octamethylcyclotetrasiloxane
(D4)2 is a colorless,
volatile liquid with a vapor pressure of 1 mm at 25°C (Flanningam,
1986
) and a water solubility of ~50 ppb (Varaprath et al., 1996). It is an industrial chemical of significant commercial importance with
uses as an intermediate in the manufacturing of polydimethylsiloxane polymers for applications such as sealants, molded rubber products, fabric coatings, electrical components, and in personal care products such as antiperspirants, shampoos, and skin creams.
The growing use of D4 in commercial products and
consumer applications prompted the Inter-Agency Testing Committee of
the Environmental Protection Agency to include
D4 on the 15th list of "Priority Chemicals"
(Inter-Agency Testing Committee, 1988), recommending that
D4 be considered for chemical and environmental fate/effects testing. As part of the chemical fate testing, several physical property determinations such as water solubility,
octanol/water partition coefficient, and Henry's Law constant
(Hamelink et al., 1996) have been made. In addition, several
biodegradation studies were undertaken: specifically, pharmacokinetic
investigations of D4 distribution, absorption,
and metabolism. The present study is concerned with the metabolism
aspect of the pharmacokinetic investigations and the identification of
essentially all major metabolites of D4 collected
from rat urine after exposure to
[14C]D4.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Instrumentation/Reagents. Radioactivity measurements were made using a liquid scintillation counter (model no. 2500 TR; Packard Instrument Co., Meriden, CT). HPLC analyses were performed with a Hewlett-Packard 1050 liquid chromatograph equipped with an HP Autosampler (model no. 79855A; Hewlett-Packard, Palo Alto, CA) and a Radiomatic detector (model no. 515 TR) from Packard. The detector was installed with a 500-µl flow liquid cell. HPLC conditions with 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 60 min. A C18 Ultima column (4.6 × 250 cm and 5 µm; Alltech Associates, Inc., 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 1:3. For samples that were not radiolabeled, an HPLC system (Hewlett-Packard) equipped with a refractive index detector (Hewlett-Packard) was used. HPLC fractions were collected using an automated fraction collector (model no. Foxy 200; Isco, Lincoln, NE) coupled to HPLC systems. Acetonitrile was purchased from Fisher Scientific and deionized water was generated using Ultrapure Water Systems (Millipore). For liquid scintillation counting, the liquid scintillants Hionic-Fluor and Ultima-Gold obtained from Packard Instrument Co. were used.
Gas chromatography-mass spectrometry (GC-MS) was performed using a Hewlett-Packard 5890 Series II Gas Chromatograph, coupled with either an HP 5989A Mass Spectrometer or an HP 5970 Series Mass Selective Detector. Both GC-MS systems were equipped with HP 7673 GC/SFC (supercritical fluid chromatography) injectors and electronic pressure control units. Data analyses were performed using a Windows-based Chem Station. GC-MS conditions were as follows: MS source temperature, 200°C; MS quadrupole temperature, 100°C (for MS 5989); detector temperature, 280°C. Oven: initial temperature, 70°C; initial time, 3 min; heating rate, 20°C/min; final temperature, 210°C; injection port, 250°C. Inlet flow settings: Helium pressure, 15 kPa at 70°C; constant flow, ON; GC column, HP-5 (5% phenylmethylsilicone); column length, 30 m; column i.d., 25 mm; film thickness, 0.25 µm. Samples or reagents were mixed with either a VWR vortex mixer (Scientific Industries Inc., Bohemia, NY) or a horizontal platform shaker (Eberbach Corporation, Ann Arbor, MI). [14C]D4 used in animal exposures was custom synthesized at Wizard Laboratories (West Sacramento, CA) and randomly labeled. The specific activity of the [14C]D4 was 2 mCi/mmol. Purity as determined by GC and HPLC was ~99%.Sample Collection.
Urine samples used in the metabolite investigation came from two
studies. In one study, male and female F-344 rats (eight animals),
CDF(F-344)/CrlBr, approximately 7 to 10 weeks of age were administered
[14C]D4 (Varaprath, 1999) as an
emulsion i.v. via tail vein injection. The animals were purchased from
Charles River Laboratories (Kingston, NY) and Charles River Canada Inc.
(Constant, Quebec, Canada). Each animal received a nominal dose
of 70 mg/kg D4. The
[14C]D4 emulsion, diluted with
unlabeled D4 to achieve the desired radioactivity
(58 µCi), was prepared in saline containing Emulphor EL-620/L.
Urine samples were collected at several time points (6, 12, 24, 48, and
72 h) after exposure. The 12-h samples from all animals were
pooled and centrifuged before analysis.
80°C until use. For the metabolite investigation in urine, i.v. was
chosen because it allows for a direct delivery of the test material,
by-passing any absorption barriers.
Concentration of samples (urine or HPLC fractions of the
metabolites) was carried out using the Speed Vac system consisting of
the following components: refrigerated condensation trap, model RT 100;
chemical trap, model SC 120; rotary vacuum pump, model VP 100; Speed
Vac concentrator, model SC 100). The system was purchased from Savant
Instruments, Inc., (Farmingdale, NY).
Extraction of Metabolites.
In metabolism studies it is desirable that the metabolites are
completely extracted into an organic solvent for structural elucidation
by GC-MS or other techniques. We have demonstrated in our previous work
(Varaprath et al., 1998) that tetrahydrofuran (THF) is an extremely
efficient solvent for extraction of urinary metabolites of
D4. The extraction efficiency was estimated to be
>95%, hence urine samples were subjected to THF extraction. It should
be pointed out that although THF is completely miscible with water, it
did form a separate layer in a urinary matrix due to the presence of
dissolved salts. Aliquots (200-500 µl) of the clear urine samples
were extracted repeatedly with THF (1- to 2-ml aliquots) until no
significant amount of radioactivity was left in the urine residues. The
combined extracts were treated with anhydrous
MgSO4 to remove water, vortex mixed (4-5 min), and centrifuged (5 min) to obtain clear, dry THF extracts. The extracts
were then concentrated to volumes of about 100- to 200-µl, using either a rotary evaporator (5°C/20 mm Hg aspirator pressure) or
by gently blowing nitrogen via Pasteur pipette along the sides of the
container vials. It was shown by HPLC analysis that the extraction and
concentration processes do not alter the metabolite profile or composition.
Derivatization.
Being water soluble and polar, urinary metabolites of
D4 were expected to have silanol structures with
multiple hydroxy functionality. Although silanols such as
dimethylsilanediol have been directly analyzed by GC-MS (Varaprath et
al., 1995), silanols with >2 OH groups per Si do not survive the GC
conditions and do not elute as such in GC. For this reason, the silanol
functions of the metabolites were protected before GC-MS analysis using
bis(trimethylsilyl)trifluoroacetamide (BSTFA), a trimethylsilylating
agent. The reaction with BSTFA was quite rapid and took place under
neutral conditions. The structural integrity of the silanol metabolites
was expected to be preserved in such a mild environment. The
dried and concentrated THF extracts were treated with equivalent
amounts (v/v) of BSTFA purchased from Petrarch (now United Chemical
Technologies, Inc., Bristol, PA), 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
hexamethyldisiloxane (MM, 99.9% pure, obtained by spinning band
distillation) was carried out in a similar way, except that a catalytic
amount of hydrochloric acid also was added. This latter reagent was
used to derivatize the metabolite dimethylsilanediol (containing single
silicon species) only, because metabolites containing more than one
silicon atom will undergo hydrolysis under acidic conditions.
Aqueous Silanol Functionality Test (ASFT; Mahone et al., 1983) of
Urine Samples.
A urine sample (500 µl) was placed in a 4-oz Teflon bottle with a
screw cap, and 80 ml of 10% (w/v) hydrochloric acid was added. The
bottle was tightly closed and shaken for 72 h using a wrist action
mechanical shaker at ambient temperature. One milliliter of very high
purity (99.9%) MM (11) was added to the bottle and shaking was
continued for 48 h. The contents were then allowed to settle for
about 10 min. First, the bottom aqueous phase was removed as completely
as possible using a 100-ml syringe. The residual aqueous phase, along
with the MM layer, was then collected into a 7-ml vial and centrifuged
for 5 min. The clear MM layer was collected for analysis by GC-MS or HPLC.
[14C]Methylsilanetriol [MeSi(OH)3, A]. This material was received from General Electric Company (GE). It was stored as an aqueous solution at a 650-ppm concentration. The radioactivity of the aqueous solution was determined to be 0.0071 mCi/ml. In HPLC, the material eluted at ~3.4 min.
[14C]Dimethylsilanediol [Me2Si(OH)2 or Monomerdiol, D]. One hundred milliliters of deionized water was placed in a clean 4-oz. Teflon bottle and 15 µl of [14C]dimethyldimethoxysilane (neat liquid, 98.3% pure by GC, specific activity 2.4 mCi/mmol; custom synthesized at Wizard Laboratories) was added. The bottle was closed tightly with a Teflon screw cap and the contents were stirred using a magnetic stir bar for 2 h. Based on the expected quantitative transformation to dimethylsilanediol, the final concentration of the dimethylsilanediol was calculated to be ~100 ppm. In HPLC, this component eluted at ~13.4 min.
[14C]Tetramethyldisiloxane-1,3-diol [HO-(Me2SiO)2-H or Dimerdiol, F]. Twenty microliters (neat liquid) of [14C]dimethyldimethoxysilane (specific activity 2.4 mCi/mmol) was placed in a plastic centrifuge tube and 1 ml of deionized water was added. The tube was capped, vortex mixed for about 5 min, and set aside at room temperature. The sample was analyzed by HPLC under the usual conditions and the component eluted at ~31.7 min. At equilibrium, the dimerdiol (based on relative peak area of C-14 to the parent monomerdiol) accounted for about 22%.
[14C]Hexamethyltrisiloxane-1,5-diol [HO(SiMe2-O)3-H or Trimerdiol, G]. One hundred microliters of the equilibrium mixture containing 22% [14C]HOSiMe2-O-SiMe2OH and 78% [14C]Me2Si(OH)2 was mixed with 200 µl of deionized water and 700 µl of Dow Corning (Midland, MI) Z-6329 Silane [Me2Si(OMe)2]. The contents were placed in a plastic centrifuge tube and vortex mixed at room temperature for 3 min and then shaken at 65°C for 24 h in a water bath. An HPLC radiochromatogram indicated a mixture containing dimer- and trimerdiol (70:30%, respectively). The trimerdiol eluted at 36.5 min.
[14C]Trimethyldisiloxane-1,3,3,-triol [(HO)2SiMe-O-SiMe2OH, E] Method A. One hundred microliters of the 20,000-ppm aqueous solution of unlabeled Me2Si(OH)2 was mixed with 50 µl of a 650-ppm aqueous solution of [14C]MeSi(OH)3 (radioactivity 0.0071 mCi/ml). The ingredients were placed in a 7-ml-capacity glass vial and shaken at 50°C for 3 h. The HPLC radiochromatogram showed that the desired product eluted at 28.3 min. [Note: the 20,000 ppm aqueous solution of Me2Si(OH)2 in turn was prepared by hydrolyzing 600 µl of Me2Si(OMe)2 with 20 ml of deionized water in a high-density polyethylene bottle at room temperature over a 3-h period.]
[14C]Trimethyldisiloxane-1,3,3,-triol [(HO)2SiMe-O-SiMe2OH, E] Method B. Three hundred microliters of the 100-ppm [14C]Me2Si(OH)2 aqueous solution was mixed with 300 µl of a 5000-ppm aqueous solution of unlabeled MeSi(OH)3. The ingredients were placed in a 7-ml-capacity glass vial, then shaken at 45°C for 1 h. The HPLC radiochromatogram showed the product eluting at 28.2 min. [Note: the 5000-ppm aqueous solution of MeSi(OH)3 in turn was prepared by hydrolyzing 757 µl of MeSi(OMe)3 with 100 ml of deionized water in a 4-oz. Teflon bottle at room temperature overnight.]
[14C]Dimethyldisiloxane-1,1,3,3,-tetrol [(HO)2SiMe-O-SiMe(OH)2, C]. One hundred microliters of a 650-ppm aqueous solution of [14C]MeSi(OH)3 (specific activity 0.0071 mCi/ml) was mixed with 200 µl of a 5000-ppm aqueous solution of unlabeled MeSi(OH)3. The ingredients were shaken at 50°C for 3 h in a 7-ml-capacity glass vial. To increase the amount of the desired material, an additional 100 µl of 10,000-ppm aqueous unlabeled MeSi(OH)3 was added and the mixture was heated at 50°C for 1.25 h. An HPLC radiochromatogram indicated that the desired product eluted at 7.5 min.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Metabolite Profile.
Of the various biological matrices, urine was chosen as the
investigative medium because it is known to contain mostly metabolites. A metabolite profile showing the number and relative amounts of various
biotransformation products generated from the parent material can be
readily obtained if the metabolites are radiolabeled. Accordingly, rats
were administered [14C]labeled D4
(Varaprath, 1999); urine samples were collected for analysis.
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Identification of Major Metabolites (A): MeSi(OH)3 and (D): Me2Si(OH)2. Structural assignments were made from GC-MS analyses of the trimethylsilylated THF extracts of the metabolites from urine. Tentative assignments of the structures were also made from HPLC analysis. HPLC retention time comparisons were made of the metabolites in urine with that of the synthetic 14C-labeled standards3. Control urine samples collected from rats not exposed to [14C]D4 were also subjected to extraction, derivatization, and GC-MS analysis. Experiments with controls were especially critical in analyzing silicones to verify the absence of the potential metabolites in solvents and derivatization agents.
Metabolite A. MeSi(OH)3
(Methylsilanetriol).
The clue to the presence of this metabolite came from the application
of an analytical procedure referred to as the ASFT (see Materials
and Methods; Varaprath et al., 1998). This is a novel method developed for the characterization of waterborne organosilicon substances. The technique is capable of analyzing waterborne
organosilicon materials in the parts per million range. Stated briefly,
in this method, all siloxane species are reduced to single
silicon-containing species with -OH functionalities. Then in a
subsequent step, the -OH functionalities are protected with
trimethylsilyl groups to enable elution in GC or GC-MS for
identification. Following this method, the urine sample was first
subjected to acid digestion at ambient temperature with 10%
(w/v) hydrochloric acid, during which the various metabolites
present in urine underwent Si-O bond cleavage, producing three
hydrolysis products, each containing a single silicon atom and two to
four hydroxy groups (Fig. 2).
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Metabolite D, Me2Si(OH)2
(Monomerdiol).
Significant amounts of MDM in the ASFT (Figs. 3 and 4) of rat urine
containing D4 metabolites strongly suggested
precursors of the structure
HO-(Me2SiO)x-H, among
others. Of these, the presence of HO-(Me2SiO)-H
(dimethylsilanediol or monomerdiol) was confirmed by HPLC
radiochromatogram comparison with an authentic sample of
[14C]labeled material. The HPLC retention time (13.2 min)
of the synthetic standard was identical with that of a peak found in urine. The standard 14C-labeled
Me2Si(OH)2 was synthesized
by hydrolyzing
[14C]Me2Si(OMe)2
(Varaprath, 1999) under neutral conditions with deionized water, and
its formation was confirmed by direct GC-MS (electron impact
ionization/selected ion monitoring mode) analysis (m/z 77, M-15) on a 10-ppm dilute solution in THF
(Varaprath et al., 1995).
Identification of Minor Metabolites: Metabolites B: (OH)3 Si-O-Si(OH)Me2 and C: MeSi(OH)2-O-Si(OH)2Me. The HPLC retention times of B and C lie between those of A (3OH functions per Si) and D (2OH functions per Si), but much closer to A than D. The very low retention times of these metabolites would indicate the prevalence of OH functions, with a lesser number of methyl groups per silicon atom. An ion-extract chromatogram of the BSTFA-derivatized THF extract of the test urine sample showed the presence of three components (retention times: 8.98, 9.09, and 9.18 min), each with a mass m/z of 443. The mass fragmentation patterns were quite similar, which suggested that they are structural isomers. The component with the retention time 9.09 min was determined to be tetradecamethylpentasiloxane (an impurity in BSTFA) by comparison to a standard (obtained by fractionation of a mixture containing linear siloxanes). Several structural possibilities existed for the other two components that pertain to mass 443. However, taking into consideration both the HPLC and GC-MS data, the following structures were tentatively assigned for the trimethylsilyl (TMS) derivatives eluting at 8.98 and 9.18 min, respectively: Me2Si(OTMS)-O-Si(OTMS)3 and MeSi(OTMS)2-O-Si(OTMS)2Me. These structures indicated the presence of corresponding silanols Me2Si(OH)-O-Si(OH)3 and MeSi(OH)2-O-Si(OH)2Me as metabolites in urine. Because the structure with the silicic acid backbone [that is, Me2Si(OH)-O- Si(OH)3] was expected to be more polar, this was assigned to component B in the HPLC radiochromatogram, and the other structure [MeSi(OH)2-O-Si(OH)2Me] to C.
An authentic 14C-labeled sample of MeSi(OH)2-O-Si(OH)2Me was synthesized by condensation of [14C]MeSi(OH)3 with unlabeled MeSi(OH)3. The latter was added to increase the concentration of the reactant and facilitate the equilibration to dimerization. The reaction was also conducted at a slightly elevated reaction temperature (40-50°C) to rapidly attain equilibrium. GC-MS of the TMS derivative from this synthetic material extracted into THF was found to match both in retention time and mass spectral characteristics to the metabolite component eluting at 9.18 min (m/z = 443, 355, 281, 221, 147, and 73). Also, this synthetic [14C]MeSi(OH)2-O-Si(OH)2Me had the same HPLC retention time as the component C present in urine samples. The synthesis of Me2Si(OH)-O-Si(OH)3 was attempted by the reaction of an aqueous solution of silicic acid [generated from the hydrolysis of Si(OMe3)4] with [14C]Me2Si(OH)2. Only trace amounts of the desired product could be obtained but the HPLC retention time of the synthetic material matched that of metabolite B, and the mass spectral fragmentation patterns of their TMS derivatives were identical and nearly identical with that of the TMS derivative of metabolite C (see above).Minor Metabolites, F: HO-(Me2SiO)2-H (Tetramethyldisiloxane-1,3-diol or Dimerdiol) and G: HO-(Me2SiO)3-H (Hexamethyltrisiloxane-1,5-diol or Trimerdiol). As noted earlier, significant amounts of MDM in the ASFT (Figs. 3 and 4) of rat urine containing D4 metabolites indicated the presence of several structures, including the type HO-(Me2SiO)x-H. Of these, the presence of HO-(Me2SiO)-H (D) has already been clearly demonstrated. The presence of HO-(Me2SiO)2-H and HO-(Me2SiO)3-H as metabolites of D4 in urine was also established (as in the case of D), by generating authentic 14C-labeled materials and making HPLC comparisons.
A simple way to generate these materials has been found. It is known that at concentrations of just 2% in aqueous solutions, dimethylsilanediol undergoes a self-condensation reaction to form the dimer (Varaprath et al., 1995). We have taken advantage of this ready
dimerization tendency by hydrolyzing
[14C]Me2Si(OMe)2
(20 µl) with 1 ml of deionized water to a achieve a 2% (v/v)
solution of
[14C]Me2Si(OH)2,
promoting self-condensation. To accelerate the equilibration reaction
to form the dimerdiol, the reaction mixture was then gently shaken at a
slightly elevated temperature (40-50°C). The progress of the
reaction was monitored by HPLC analysis.
By increasing the concentration of the reaction mixture through added
Me2Si(OMe)2 (labeled or
unlabeled) and continuing the heating, additional condensation to form
the trimerdiol (G) was achieved. From the HPLC analysis,
the retention times for the dimerdiol and trimerdiol standards
generated were determined to be 31.7 and 36.5 min. The two components
in the test urine that eluted at essentially identical retention times
were therefore assigned the corresponding structures. GC-MS analysis of
their TMS derivatives showed masses m/z 295 (M-15), 207, 103, and 73 and 369 (M-15), 281, 147, and 73, respectively, as expected for the products MD2M
and dodecamethylpentasiloxane. The retention times 6.51 and 8.08 min, respectively, and mass spectral patterns of these TMS derivatives
also matched commercially available standards.
Metabolite E: Me(OH)2Si-O-Si(OH)Me2. The HPLC retention time of metabolite E lies between that of dimethylsilanediol (D) and tetramethyldisiloxane-1,3-diol (dimerdiol, F), but much closer to the latter. Therefore, the metabolite must have an intermediate polarity, suggesting the possibility of an oxidized dimerdiol structure, [Me(OH)2Si-O-Si(OH)Me2], for this metabolite, containing three OH functions per molecule.
To conclusively prove the structure, the 14C-labeled standard was synthesized by two different routes. In one, [14C]MeSi(OH)3 was allowed to react with unlabeled Me2Si(OH)2 and in the other, unlabeled MeSi(OH)3 was allowed to react with [14C]Me2Si(OH)2. The resulting products from both of the routes had similar retention times in HPLC: 28.3 and 28.2 min, respectively, and they matched the component present in urine. GC-MS analyses of the TMS derivative from the synthetic material also showed similarities in retention time (7.93 min) and mass spectral characteristics (m/z = 369, 281, 147, and 73) to the component present in the urine.| |
Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Organosilane and siloxane compounds are known to
undergo metabolic transformation. The metabolic fates of several
silicon-containing compounds have been reported. Fessenden and Ahlfors
(1967) examined urinary metabolites of silacarbamates, viz,
bis(hydroxymethyl)dimethylsilane dicarbamate
(CH3)2Si(CH2OCONH2)2,
(hydroxymethyl)-dimethyl-n-propylsilanecarbamate CH3CH2CH2Si(CH3)2CH2OCONH2,
and bis(hydroxymethy)-di-n-propylsilanedicarbamate (CH3CH2CH2)2Si(CH2OCONH2)2
after oral administration to rats. The formation of oxidative
dealkylation (silanols) and oxidative hydroxylation products was observed.
In dealing with any metabolism work involving silicones, it is
important to be cognizant of potential problems. The procedures normally used in identification of organic metabolites may not be
directly applicable to silicones due to significant differences in
chemical properties between the two. Organic metabolites can often
tolerate harsh workup procedures. However, in the case of silicones,
milder workup procedures should be used. Acids and bases must be
avoided, because rearrangements, self-condensation, and isomerization
are common occurrences under these conditions. These phenomena are
especially pronounced if the metabolites contain hydroxy functions. The
extreme affinity of silicon for oxygen is the cause of several
of the rearrangement reactions. Functional groups such as -OH, -COOH,
and -CH2OH are commonly seen in organic drug
metabolites. If such functionalities are formed from siloxane metabolism, they can potentially undergo rearrangement with migration of the Si atom from carbon to oxygen (Brook and Gilman, 1955; Brook and
Iachia, 1961). In addition, background levels of silicone species in
solvents used for extraction, or reagents used for metabolite
derivatization, may also contribute to artifacts (Fessenden and
Hartman, 1970).
In conclusion, the major metabolites of octamethylcyclotetrasiloxane (D4) in rat urine have been identified. The methods used were quite specific, and minimize ambiguities in the structural assignments. Simple procedures were developed to synthesize 14C-labeled metabolite standards, which enabled direct HPLC comparisons with urinary components. This methodology greatly facilitated structural identification. The process also used the optimum solvent (THF) to extract the metabolites (essentially quantitatively) and a mild derivatization agent (BSTFA) to permit identification using GC-MS. The metabolites identified have also clearly established that some demethylation occurs at the silicon-methyl bond.
Mechanistic Pathways for Metabolite Formation.
The mechanistic pathways for the formation of the various metabolites
are not clearly understood. However, possible pathways for their
generation are speculated and presented in Fig.
5, where it is suggested that
intermediate 9 can be the precursor to most of the
metabolites A to G. It is known that such an
intermediate is generated in the atmospheric oxidation of
D4 (Sommerlade et al., 1993). However, to our
knowledge, there is no precedent for direct oxidation of a
Si-CH3 group to an Si-OH function in drug metabolism. We therefore propose the formation of an intermediate Si-CH2OH group (structure 8), which
is well precedented to rearrange to Si-O-CH3
(10); The latter can hydrolyze easily to methanol and
Si-OH. The sequence Si-CH2OH 
-Si-CHO -Si-OH resulting from continued oxidation and hydrolysis reactions is
also plausible.
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), whereas hydrolysis may or may not be enzyme-mediated. It is not necessary that metabolites containing fewer silicon atoms than the
parent D4 [such as
Me2Si(OH)2] be formed only
by sequential hydrolysis of higher homologs. A one-step
mechanism involving simultaneous cleavage of two Si-O bonds can also be
envisioned to form
Me2Si(OH)2.
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Acknowledgments |
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We thank Joan McMahon, Steve Crofoot, and Brad Hubbell for exposing animals to D4 and collecting the rat urine samples needed for metabolite identification.
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Footnotes |
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Received November 6, 1998; accepted May 27, 1999.
1 Present address: SmithKline Beecham, King of Prussia, PA.
This work was supported in part by the Silicones Environmental, Health and Safety Council of North America.
3 HPLC and GC-MS of standards were not included in this manuscript. They will be available on request.
Send reprint requests to: Sudarsanan Varaprath, Ph.D., Senior Research Chemist, Dow Corning Corporation, 2200 W. Salzburg Road, Auburn, MI 48686-0994. E-mail: sudarsanan.varaprath{at}dowcorning.com
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Abbreviations |
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Abbreviations used are: MM, hexamethyldisiloxane; MDM, octamethyltrisiloxane; MD2M, decamethyltetrasiloxane; M3T, methyltris(trimethylsiloxy)silane; M4Q, tetrakis(trimethylsiloxy)silane; D4, octamethylcyclotetrasiloxane; GC-MS, gas chromatography-mass spectrometry; THF, tetrahydrofuran; BSTFA, bis(trimethylsilyl)trifluoroacetamide; ASFT, Aqueous Silanol Functionality Test; The abbreviations are based on the General Electric's siloxane notation [Hurd CB (1946) Studies on siloxanes. 1. The specific volume and viscosity in relation to temperature and constitution. J Am Chem Soc 68:364] which is as follows, M, Me3SiO1/2; D, -Me2SiO2/2; T, MeSiO3/2; Q, SiO4/2.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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