Introduction

Abstract Introduction Results Discussion References

CHO² {7-oxabicyclo[4.1.0]heptane(9CI,8CI); CAS registry no. 286-20-4} is present in natural products and has a wide range of uses in industry. The compound is primarily used as a raw material in the organic synthesis of various kinds of alicyclic chemical intermediates for pesticide, pharmaceutical, perfumery, and dyestuff manufacture. CHO is also used as a monomer in photoreactive polymerizations. According to the 1983 National Occupational Exposure Survey, 2874 workers were potentially exposed to CHO.

CHO can be formed by cytochrome P450-mediated oxidative metabolism of cyclohexene (1, 2). After its formation, the oxide can undergo hydrolysis by epoxide hydrolase or conjugation with GSH (3). Although CHO seems to be relatively nontoxic when given orally to rats (LD₅₀: 105 g/kg), it does cause a significant number of biological effects. For example, CHO is a noncompetitive inhibitor of microsomal, but not cytosolic, epoxide hydrolase in mammalian liver, kidney, lung, intestine, and skin (4, 5). It has also been shown to reduce hepatic GSH levels by at least 10% (2). This interaction with phase II enzyme systems creates the potential for interaction with other chemicals. For example, CHO has been shown to increase the in vivo hepatotoxicity of 1,1-dichloroethylene (6) and to alter the toxicity of bromobenzene in isolated hepatocytes (7). Furthermore, CHO significantly increases the DNA binding of dibenzo(a,e)fluoranthene in mouse and rat liver microsomal preparations (8). These chemical-chemical interactions are likely mediated by CHO-associated reductions of epoxide hydrolase activity or GSH content. However, this depletion in cellular oxidative defense systems could result in organ specific toxicity. Therefore, a more extensive toxicological evaluation of CHO is warranted.

At present, very little information is available on the disposition and metabolism of CHO. Therefore, the present study was designed to provide insight into the biological fate of CHO in animals. The objectives of this study were to examine the absorption, distribution, metabolism, and excretion of CHO after oral, intravenous, and dermal exposure in female B6C3F₁ mice and Fischer 344 rats. Furthermore, a toxicological study was performed after both oral and topical administrations.

Materials and Methods

Chemicals.  [¹⁴C]CHO was obtained from New England Nuclear (Boston, MA). Radiochemical purity was >98%, as determined by HPLC radiochemical analysis. The specific activity was reported by New England Nuclear to be 3.1 mCi/mmol. Unlabeled CHO (vapor pressure: 12 mbar) was obtained from Fluka Chemika Biochemika (Buchs, Switzerland), and the chemical purity of the compound was determined to be >99% by GLC using FID. All other reagents used in these experiments were either of analytical or HPLC grade.

Animal Studies. Animals.  Male JVC F-344 rats (175-250 g) were obtained from Hilltop Lab Animals, Inc. (Scottsdale, PA). Female B6C3F₁ mice (21-27 g) were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Upon arrival, animals were acclimated for 5-7 days in a temperature-controlled 12-hr light/dark cycle facility before any treatment. Animals were provided food (Teklad 4% Mouse-Rat Diet, Teklad, Madison, WI) and water ad libitum. Because the metabolism and disposition of CHO could be both sex- and species-dependent, male Fischer 344 rats and female B6C3F₁ mice were used in these experiments.

Preliminary Studies.  For each route of administration, rats and mice were housed in air-tight glass metabolism cages and exhaled radiolabel collected for at least 48 hr. [¹⁴C]CHO equivalents associated with exhaled organics were trapped with 2-methoxyethyl ether (Mallinckrodt Chemical, Paris, KY), and [¹⁴C]CO₂ was trapped with CarboSorb (Packard, Meriden, CT)/ethylene glycol (Mallinckrodt Chemical), 2:1 (v/v). Trapping solvents were changed and measured for total radioactivity by scintillation counting at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, and 48 hr after administration of [¹⁴C]CHO.

Intravenous Administration.  To determine pharmacokinetic parameters, [¹⁴C]CHO (50 mg/kg, 120 µCi/ml) in saline was administered to male JVC F-344 rats (>5 sec). The injection was followed by an equal volume of normal saline to flush the cannula. The dosing solution was prepared just before use. After compound administration, animals were then placed into Nalgene metabolism cages to allow collection of urine (6, 12, 24, and 48 hr) and feces (24 and 48 hr) throughout the 48-hr experiment. Blood samples (250 µl) from male JVC F-344 rats were collected via the jugular cannula at 0, 0.05, 0.083, 0.167, 0.33, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 8, 12, and 24 hr, and analyzed for CHO by GLC-FID. At the end of 48 hr, animals were subjected to CO₂ euthanasia. Blood samples were centrifuged, and the plasma was stored at 20°C until analyzed for CHO and its metabolites. All plasma samples were analyzed within 48-72 hr of collection. In addition, various tissue samples (brain, subcutaneous fat, testicular fat, heart, kidneys, large intestine, small intestine, stomach, liver, lungs, muscle, skin, testes, ovaries, and gastrointestinal contents) were harvested from rats (48 hr) and stored at 70°C until analyzed for total radioactivity by tissue oxidation as described by Winter and Sipes (9). Body composition estimates of 11% for adipose tissue, 9% for blood, 50% for muscle, and 16% for skin were used (10, 11).

Oral Administration.  [¹⁴C]CHO (10 and 100 mg/kg) in corn oil was administered to the animals by oral gavage, four rats per dose and four mice per dose per time point. The dosing solution was prepared just before use. All animals received ~50 µCi/kg of the radiolabeled compound. Animals were then placed into Nalgene metabolism cages to allow collection of urine (6, 12, 24, and 48 hr) and feces (24 and 48 hr) throughout the 48-hr experiment. Blood samples (250 µl) from JVC F-344 rats were collected via the jugular cannula at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 24, and 48 hr. Mice were killed at each time point by CO₂ inhalation. Blood was collected immediately from the posterior vena cava into a heparinized syringe, and the major tissues were harvested. ¹⁴C-equivalents present in the tissues were determined by tissue oxidation.

Dermal Administration.  The in vivo percutaneous absorption of CHO after topical application was determined using the method of Winter and Sipes (9). Eighteen hours before CHO exposure, male JVC F-344 rats and female B6C3F₁ mice were anesthetized, backs shaven, and aluminum skin traps were affixed onto the animals with cyanoacrylate adhesive. The traps circumscribe an area of 8.4 cm² (rat) and 0.8 cm² (mouse) on the skin, and were affixed to the hairless back over the midthoracic region. The skin traps consist of an outer aluminum casing with an inner stainless-steel basket (~0.6-1.3 cm above the skin) that contains activated charcoal (2.6 and 20 g, 20-40 mesh). After a recovery period of at least 16 hr, [¹⁴C]CHO (60 mg/kg, 50 µCi/kg) in acetone (100 µl for the rat and 10 µl for the mouse) was applied using a blunted needle inserted through the charcoal layer of the skin trap, four rats total and four mice per time point. A needle guide and a depth gauge were used to ensure that the tip of the needle was just above the surface of the skin, but below the charcoal adsorbent layer. The dosing solution was prepared just before use.

Plasma and pH Stability of CHO.  After oral administration, CHO would be subjected to low pH in the stomach, which could facilitate the hydrolysis of the epoxide moiety and decrease the absorption of intact CHO. Therefore, a study was conducted to determine the stability of CHO in buffers of different pH. CHO (480 µg/ml) was incubated at 37°C for 4 hr in solutions that ranged from pH 1 to 7. After incubation, samples were extracted with ethyl acetate and analyzed for CHO and cyclohexane-1,2-diol using GLC-FID. Furthermore, because of the possible instability of CHO in plasma, studies were also performed to determine if CHO was stable in plasma. Plasma (100 µl) was spiked with CHO (24 µg/100 µl) and incubated at 37°C for 1, 2, 4, 8, 12, and 24 hr. At the end of the incubation period, samples were extracted and analyzed for CHO and cyclohexane-1,2-diol.

Toxicity Study.  After a 14-day quarantine period, animals were assigned at random to treatment and control groups. All male mice were housed individually, and female mice and rats were housed in groups of five animals per cage. For oral gavage studies, animals were treated with CHO (100, 50, 25, 12.5, and 6.25 mg/kg) or its vehicle (0.5% methyl cellulose), five animals per sex per species received CHO for 28 days (5 days/week). Treatment was not performed on weekends or holidays, and animals were dosed at least two consecutive days before the terminal sacrifice. For dermal studies, animals were exposed topically to CHO (60, 30, 15, 7.5, and 3.75 mg/kg) or its vehicle (acetone), five animals per sex per species. Each dose concentration remained constant, and the volume (2 ml/kg for rats, 0.5 ml/kg for mice) was adjusted weekly to maintain the appropriate mg/kg mean body weight exposure. Dosing solutions were prepared every 2 weeks. Analysis of dosing solutions was performed by capillary GC and found to within 10% of the target concentration. Animals were weighed on the first day of CHO administration, once each week and at euthanasia. After euthanasia, organ weights (liver, thymus, right kidney, right testicle, heart, and lungs) were determined for all animals. Histopathological evaluation was performed on heart, ovary, forestomach (gavage-dosed animals), and skin (topically dosed animals). These organs were evaluated because they demonstrated possible lesions of interest.

Data Analysis.  The plasma concentration-time data after intravenous bolus dosing were analyzed by both compartmental and noncompartmental methods. Disposition parameter values best describing a linear multicompartmental model were determined from nonlinear regression analysis using a weighting scheme of 1/Y² (12). The most appropriate model was determined with application of the F test (13). The parameters of the model were used to calculate values for CL_s, V_ss, t_1/2, and MRT. The average plasma concentration-time data were also fit to the appropriate model to provide a graphical display of the data.

Analytical Methods. GLC Analysis of CHO and Its Metabolites. The GLC analysis of CHO and its metabolites used in these studies has been described in detail by Bao et al. (15). Briefly, to extract CHO and cyclohexane-1,2-diol, an equal volume of ethyl acetate/acetonitrile [7:3 (v/v)] was added to plasma and urine samples. After twice vortexing for 30 sec, samples were centrifuged and the organic extracts removed and pooled. An aliquot of the pooled organic samples (1 µl) was injected onto a HP-FFAP fused silica capillary column (0.52-µm film thickness, 0.32 mm diameter, 25 m; Hewlett-Packard, Wilmington, DE) installed in a Hewlett-Packard Series II Plus 5890 with an FID. The oven temperature was initially maintained at 50°C for 2 min, then increased at 7°C/min for the next 21 min to a final temperature of 200°C, and maintained at 200°C for 3 min. The detector and injector temperatures were 250°C and 200°C, respectively. Helium was used as the carrier gas, and air was used as the auxiliary gas (60 ml/min). CHO, trans-cyclohexane-1,2-diol, and cyclohexanol standards were extracted from spiked plasma and urine, and the resulting standard curve was used to calculate the plasma and urine concentration of these analytes. The limit of detection for CHO, trans-cyclohexane-1,2-diol, and cyclohexanol was 1 µg/ml.

HPLC Analysis of CHO and Its Metabolites in Urine.  For analysis of urinary metabolites, acetonitrile:isopropanol [1:1 (v/v)] was added to samples in a 1:1 ratio. Samples were vortexed and then centrifuged to remove any precipitate. Prepared urine samples (100 µl) were injected onto a 250 × 4.6 mm Zorbax CN (7 µm) column (Phenomenex, Torrance, CA) eluted with a mobile phase of water:acetonitrile:isopropanol at a flow rate of 1 ml/min with a total run time of 30 min. The gradient was 1:1 acetonitrile:isopropanol for 5 min and then changed to obtain 1:4:5 water:acetonitrile:isopropanol over a 5-min period, where it remained for another 5 min. The remainder of the run was eluted using 1:1 acetonitrile:isopropanol. The HPLC (Spectra Physics, SP 8800, San Jose, CA) was equipped with an autosampler (Thermo Separation Products-Spectra Systems AS 3000, Fremont, CA), and column effluent was monitored radiochemically using a -RAM flow-through monitor (IN/US Systems, Inc., model 2 with WinFlow, Tampa, FL). Samples were also incubated with -glucuronidase (2000 units/ml) or sulfatase (100 units/ml) for at least 24 hr and analyzed by HPLC.

Metabolite Identification Using MS and MS/MS Analysis.  For metabolite isolation and identification, pooled urine samples from the oral administration study (6, 12, 24, and 48 hr collection) were diluted with acetonitrile:isopropanol [1:1 (v/v)] and centrifuged to remove any precipitate. HPLC peaks containing radioactivity were collected and concentrated by lypholization. Concentrated fractions were diluted in 10 mM aqueous ammonium acetate:acetonitrile (1:1) and then analyzed on a Finnigan TSQ 7000 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an atmospheric pressure source. Samples were introduced into the mass spectrometer by flow injection at 0.5 ml/min. Ions with m/z values corresponding to putative metabolites were subjected to CID with argon gas and the subsequent product ion signals mass analyzed to produce a product ion mass spectrum. Logical fragmentation patterns observed in the resulting MS/MS spectrum provided further evidence as to metabolite identity.

	Results

Abstract Introduction Results Discussion References

Plasma and pH Stability Study.  CHO was found to be most stable at pH 7 for the 4 hr of incubation. Importantly, after 4 hr in buffers between pHs of 1-3, no intact CHO (parent) was detectable. In these low pH buffer solutions, CHO was completely converted to cyclohexane-1,2-diol. CHO was found to be stable for up to 2 hr in plasma incubated at 37°C. However, with time, it hydrolyzed to cyclohexane-1,2-diol and by 4 hr over 80% of CHO was hydrolyzed by the plasma. Within 8 to 12 hr, CHO was completely converted to cyclohexane-1,2-diol.

Intravenous Administration.  After intravenous administration, CHO was rapidly eliminated from the plasma and by 24 hr >70% of the dose had been excreted into the urine. Only a small percentage (~2.4% of the dose) appeared in the feces. Approximately 7% of the dose was exhaled and recovered in the organic (~4.0% of the dose) and CO₂ (~3.3% of the dose) traps. Cumulative excretion of radioactivity in exhaled organics/CO₂, urine, and feces is shown in fig. 1. Tissue disposition showed that little radioactivity was retained in the tissue 48 hr after treatment (~1.5% of the dose).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Cumulative recovery of total radioactivity, expressed as percentage of dose, after intravenous administration of [14C]CHO (50 mg/kg, 120 µCi/kg) in male JVC F-344 rats. Data represent mean ± SD (N = 3).

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View larger version (9K): [in this window] [in a new window]   Fig. 2.   Plasma concentration of CHO after intravenous administration of [14C]CHO (50 mg/kg, 120 µCi/kg) in male JVC F-344 rats. Data represent mean ± SD (N = 3).

Oral Administration Study.  After oral administration of CHO, the majority of the radiolabel (76-93%) was recovered in the urine (table 1). Cumulative recovery of radioactivity in the urine and feces is shown in fig. 3. Little ¹⁴C-equivalents were found to be eliminated via exhalation after oral administration. Radioactivity associated with the exhaled CO₂ and organic vapors was at or below background levels (~1.4% of the dose). Plasma samples from JVC F-344 rats treated with [¹⁴C]CHO (10 and 100 mg/kg po) were analyzed by GLC-FID for parent compound and metabolites. No unaltered CHO was detected in the plasma after oral administration at any time after dosing. However, cyclohexane-1,2-diol was detected in the plasma samples between 0.25 and 24 hr. Peak concentration of cyclohexane-1,2-diol in the plasma was 24 µg/ml at 6 hr for the 10 mg/kg dose and 34 µg/ml at 1 hr for the 100 mg/kg (fig. 4). Oxidation of tissue samples indicated that little radioactivity was retained in the tissues 48 hr after treatment (~0.7% of the dose).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Cumulative recovery of total radioactivity, expressed as percentage of dose, after oral administration of [14C]CHO (10 and 100 mg/kg, 50 µCi/kg) to male JVC F-344 rats (A) and female B6C3F1 mice (B). Data expressed as mean ± SD (N = 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Plasma concentration of cyclohexanediol expressed as µg/ml after oral administration of [14C]CHO (10 and 100 mg/kg) in male JVC F-344 rats. Data expressed as mean ± SD (N = 4).

Dermal Administration.  After dermal application of CHO, 91% of the radioactivity was absorbed onto the activated charcoal trap placed above the skin. Approximately 2% of the total dose was absorbed, because 1.3% and 0.1% of the radioactivity were recovered in urine and feces during the 48 hr after application. The skin at the site of application contained only 0.5% of the dose in the rat (table 1). No parent compound was detected in the plasma at any sample time. Similar results were obtained in mice; <5% of the total dose was absorbed. About 3% of the radioactivity was recovered in the urine and 1% in the feces. The majority of the radioactivity was recovered from the charcoal trap (table 1).

Metabolite Identification.  HPLC analysis of rat urine revealed three radioactive peaks associated with metabolites (R_T = 4.0, 9.0, and 11.5 min). Four metabolites were detected in the mouse urine (R_T = 2.5, 4.0, 9.0, and 11.5 min). Both rat and mouse urine samples contained a small peak corresponding to cyclohexane-1,2-diol (R_T = 4.0). Incubation of urine samples with -glucuronidase resulted in the loss of peak at R_T = 9.0, whereas the intensity of the peak corresponding to cyclohexane-1,2-diol increased. Incubation of mouse urine with sulfatase resulted in a reduction of the primary metabolite peak (R_T = 2.5), not found in rat urine, and increased the intensity of the peak corresponding to cyclohexane-1,2-diol. However, incubation of rat urine with sulfatase had no effect on the HPLC metabolite profile. To confirm the identity of these urinary metabolites, radioactive peaks were analyzed by MS.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Representative GC/MS EI spectra of the urinary metabolite cyclohexane-1,2-diol obtained from male JVC Fischer 344 rats after administration of [14C]CHO (HPLC peak B; RT = 4.0 min). MW, molecular weight.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Representative LC/MS/MS spectra of the urinary metabolite cyclohexane-1,2-diol-O-sulfate, cyclohexane-1,2-diol-O-glucuronide, and N-acetyl-S(2-hydroxycyclohexyl)-L-cysteine isolated from a pooled urine sample from female B6C3F1 mouse after oral administration of [14C]CHO. (A) Product ion spectra of [M-H] m/z 195, HPLC peak A (RT = 2.5 min). (B) Product ion spectra of [M-H] m/z 291, HPLC peak C (RT = 9.0 min). (C) Product ion spectra of [M-H] m/z 260, HPLC peak D (RT = 11.5 min). MW, molecular weight.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Metabolic scheme of CHO in Fischer 344 rats and B6C3F1 mice. GT, -glutamyltranspeptidase; APM, aminopeptidase M; NAT, N-acetyltransferase; GST, glutathione S-transferase; UDPGA, UDP-glucuronic acid; PAPS, 3-phosphoadenosine-5-phosphosulfate. (1) CHO, (2) cyclohexane-1,2-diol-O-sulfate, (3) cyclohexane-1,2-diol, (4) cyclohexane-1,2-diol-O-glucuronide, and (5) N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine.  Metabolite found only in female B6C3F1 mouse urine.

Toxicity Study. No statistically significant changes in final body weights or relative organ weights were noted in F-344 rats or B6C3F₁ mice treated orally with CHO up to 100 mg/kg (table 3) or up to 60 mg/kg when given topically (table 4). Very few gross lesions were found at necropsy, and none were considered compound related. Microscopic examination of the gross lesions demonstrated no compound-related pathological alterations. Although oral treatment with CHO appeared to increase lung weight in female F-344 rats, this measurement was not statistically significant due to a large variance between samples.

	Discussion

Abstract Introduction Results Discussion References

Several investigators have shown that CHO is mutagenic in many short-term assays. In the Ames histidine-reversion assay, CHO caused a significant increase in the number of revertants in several strains of Salmonella typhimurium (16-17). CHO produced mutations in the Luria and Delbruck's fluctuation test using Klebsiella pneumonia (18). CHO has also been shown to possess strong clastogenic activity, as well as cause sister chromatid exchange in V79 Chinese hamster cells (19). Thus, there is concern about the mutagenic/carcinogenic potential of CHO. Because CHO is labile, it is not known if CHO can reach its target tissues after in vivo exposure. Therefore, the absorption, distribution, metabolism, and excretion were characterized after oral, intravenous, and topical administration of CHO in both male F-344 rats and female B6C3F₁ mice.

Our results indicate that, after oral administration of CHO, ¹⁴C-equivalents are readily absorbed and cleared from the body. The major route of excretion is the urine. Tissue disposition showed that very little radioactivity was retained in the tissue 48 hr after oral administration. The apparent volume of distribution for CHO (~0.44 liter/kg) is relatively small and consistent, with a limited distribution into tissues. The short half-lives of CHO, 2.3 min (-phase) and 19.3 min (-phase), further support the rapid distribution and elimination of this epoxide. Rapid hydrolysis of CHO in low pH solutions, along with its rapid systemic elimination, explains the inability of our assay to detect CHO in the plasma after oral administration. Thus, after oral administration, it is unlikely that unaltered CHO reaches the systemic circulation in any appreciable amount due to its rapid hydrolysis in the gastrointestinal tract and because of it hydrolysis and conjugation by the liver.

Very little radiolabel was absorbed after topical application of [¹⁴C]CHO; only 4-5% of the dose gained access to the systemic circulation of rats and mice during the 48-hr experiment. Absorption was apparently limited by the loss of CHO from the skin by volatilization. Approximately 90% of the dose was trapped in the charcoal skin traps, whereas only 0.5% was found at the site of application. During the process of absorption, it is likely that CHO is subjected to hydrolysis/conjugation. The skin possesses both epoxide hydrolases and glutathione S-transferases (20) that could metabolize CHO to form cyclohexane-1,2-diol and S-(2-hydroxycyclohexyl)-L-GSH, respectively. Although the activities of dermal epoxide hydrolase and GSH S-transferases are ~10-fold lower than that found in the liver (20), they could certainly contribute to the biotransformation of CHO. There is no evidence that parent CHO reaches the systemic circulation, but very low levels of the cyclohexane-1,2-diol were observed in the plasma after dermal administration.

HPLC analysis of urinary samples further supports the conclusion that CHO was rapidly metabolized and eliminated from the body. Excretion of cyclohexane-1,2-diol, cyclohexane-1,2-diol-O-glucuronide, and cyclohexane-1,2-diol-O-sulfate (mouse only) indicates that CHO is readily hydrolyzed and conjugated. In addition to hydrolysis, CHO can undergo nucleophilic attack from GSH that would result in the formation of N-acetyl-S-(2-hydroxycyclohexyl)-L-GSH. In the present study, N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine (the mercapturate of cyclohexanol) was determined to be a major metabolite in the urine. Previously, two diastereoisomers of N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine were identified in the urine after the intraperitonal administration of CHO to rats (3). A proposed scheme for CHO metabolism is shown in fig. 7. Interestingly, the disposition, metabolism, and pharmacokinetic parameters of CHO are similar to those reported for CHOX in the rat (21). CHOX has a short t_1/2 (18.2 min) similar to CHO, and the major route of excretion is the urine. Furthermore, like CHO, a major urinary metabolite of CHOX was cyclohexane-1,2-diol-O-glucuronide. However, cyclohexanol and cyclohexanol-O-glucuronide were also detected in the urine of rats given CHOX. These metabolites were not present in the urine of rats or mice after CHO administration.

The results of the 28-day repeated dose dermal and oral toxicity studies in male and female F-344 rats and B6C3F₁ mice revealed no treatment-related mortality, clinical signs of toxicity, or histopathological evidence of toxicity up to the highest dose tested. Relative organ weights did not indicate any potential target organs in either sex species. These data are consistent with the demonstration that CHO is rapidly hydrolyzed and conjugated after both oral and dermal administrations. This would suggest that CHO is not absorbed intact to any great extent and any parent compound that is absorbed is nonrestrictively cleared due to its high hepatic intrinsic clearance (and high hepatic extraction ratio). On the basis of this discussion, the anticipated high hepatic intrinsic clearance of CHO would suggest that the compound undergoes substantial presystemic or first-pass hepatic clearance. Thus, only a minor amount of intact CHO reaches the blood, and even a smaller amount reaches the tissue after oral administration. The most reasonable explanation for the nontoxic effects after dermal dosing is that CHO is so volatile and is absorbed so slowly that very little of the parent compound penetrates the skin. Thus, the systemic exposure to CHO after dermal administration is extremely low, and the amount of intact parent reaching a given target organ is minimal.

In summary, results of the present study indicate that, after oral administration of CHO, ¹⁴C-equivalents are readily absorbed from the gastrointestinal tract, but not the skin. Absorption through the skin is incomplete due to loss of CHO by volatilization. Furthermore, it is unlikely that any unaltered CHO reaches the systemic circulation after oral or dermal exposure. Any CHO that would escape hydrolysis in gastric juices would be subjected to hydrolysis by epoxide hydrolase or conjugation by GSH transferase. This rapid metabolism likely lends to the nontoxic effects of CHO in the whole animal. Thus, because of the labile nature of CHO and widespread distribution of enzymes that can detoxify it, it is unlikely that parent CHO reaches its target tissues to cause toxicity after either oral or dermal administration. Furthermore, under the test conditions used in these studies, it seems that CHO is unable to reach its target tissues after in vivo administration to cause either toxicity or mutagenicity. Thus, in vitro tests that demonstrate cytotoxicity or mutagenicity should be viewed with caution when extrapolated to the in vivo situation.