Introduction

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1,2-Dibromo-2,4-dicyanobutane (BCB,¹ CAS registry no. 35691-65-7) has a wide range of industrial and commercial applications. Its primary use has been as an antimicrobial agent to inhibit the growth of slime-forming bacteria and fungi. The use of BCB in cosmetic formulations suggests potential for widespread human exposure. Interestingly, in these formulations BCB has been shown to be a weak skin sensitizing agent in man (Hausen, 1993; Tosti et al., 1995). Although no data are available in the open literature regarding the specific toxicities of BCB, its oral LD₅₀ in rats has been estimated to be 800 mg/kg. BCB is not mutagenic in Salmonella typhimurium, nor does it induce unscheduled DNA synthesis in IMR-90 human embryonic fibroblasts (Anonymous, 1994). However, in an in vitro Chinese hamster ovary cell assay, BCB produced significant frequencies of chromosome aberrations (Anonymous, 1994). Although BCB has been shown to be relatively nonmutagenic in several in vitro assays, it shares structural similarity to several halide-containing compounds, which have been shown to be carcinogenic in animal models.

BCB is structurally similar to ethylene-1,2-dibromide and 1,2-dibromopropane, two compounds that have been well characterized from both a biotransformation and carcinogenic standpoint (Guengerich, 1994; Lag et al., 1994; Van Duuren et al., 1985; Zoetemelk et al., 1986). In both instances, glutathione conjugation and oxidation play important roles in the carcinogenic potential of these compounds. Glutathione reacts directly with ethylene-1,2-dibromide to form the highly reactive episulfonium ion (Guengerich, 1994). This electrophilic species is responsible for both the mutagenicity and DNA binding associated with this compound. On the other hand, 1,2-dibromopropane is weakly mutagenic and does not directly interact with glutathione to form the episulfonium ion (Lag et al., 1994). Although the formation of this reactive metabolite has been proposed, this pathway does not seem to be a predominate route of biotransformation for 1,2-dibromopropane (Zoetemelk et al., 1986). Instead, the primary metabolic pathway of 1,2-dibromopropane is oxidation followed by glutathione conjugation.

Ethylene-1,2-dibromide and 1,2-dibromopropane differ only by a single methyl group, but their ability to participate in carcinogenic events is vastly different because of subtle differences in bioactivation. Although metabolic activation in many cases dictates the carcinogenic potential of halide-containing alkanes, very little is known about the metabolism and fate of BCB. Therefore, the primary objective of this study was to determine the absorption, distribution, biotransformation, and excretion of BCB following iv, oral, and topical exposure in the male Fischer 344 rat.

	Materials and Methods

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Chemicals. Radiolabeled [¹⁴C-1,3]BCB (9.42 mCi/mmol) was obtained from Wizard Laboratories (West Sacramento, CA). [¹⁴C-1,3]2-MGN was synthesized by reacting [¹⁴C]BCB (90 µCi, 7.0 mg) with glutathione (34.7 mg) in 1.5 ml of saline for 3 hr at 37 °C. Both compounds were greater than 99% pure as determined by HPLC radiochemical analysis and GC-MS analysis. Unlabeled BCB was obtained from Calgon Corporation (Pittsburgh, PA). Its chemical purity was determined to be greater than 99% by GC-MS analysis.

Animals. Male Fischer 344 rats (175-250 g) with and without jugular vein cannulation (JVC) were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). The animals were acclimated for 5-7 days in a temperature-controlled (25 °C) 12-hr light/12-hr dark cycle facility. The animals were provided food (Teklad 4% mouse-rat diet, Harlan Teklad, Madison, WI) and water ad libitum. The doses selected for the following animal studies were based on 1/10 (oral) and 1/100 (iv) the oral LD50 of BCB in rat (800 mg/kg). Furthermore, these doses were chosen because they were not acutely toxic.

Intravenous Administration. [¹⁴C]BCB (8 mg/kg, 120 µCi/kg), in emulphor/ethanol/saline, 3:4:12 (v/v/v) was administered via a jugular vein cannula to male Fischer 344 rats (over 5 sec). The injection (2 ml/kg) was followed by an equal volume of normal saline to flush the cannula. The animals were then placed in Nalgene metabolism cages to allow collection of urine and feces throughout the experiment. Blood samples (250 µl) were collected via the jugular cannula at selected points (0, 1, 3, 5, 7, 10, 12, 15, 20, 30, and 45 min, as well as 1, 6, 12, 24, and 48 hr) and analyzed immediately by HPLC. The withdrawn blood volume was replaced with an equal volume of saline.

Oral Administration. [¹⁴C]BCB (80 mg/kg, 100 µCi/kg) in corn oil (5 ml/kg) was administered orally by gavage to male JVC Fischer 344 rats. The animals were then placed in Nalgene metabolism cages to allow collection of urine and feces. Blood samples were collected at selected points (0, 15, 30, and 45 min, as well as 1, 2, 3, 4, 6, 8, 12, 24, and 48 hr). At the end of the 72-hr experiment, the animals were subjected to carbon dioxide euthanasia.

Topical Administration. The in vivo percutaneous absorption of [¹⁴C]BCB following topical application was determined using the method of Winter and Sipes (1993). Following adhesion of the percutaneous traps and a recovery period of at least 16 hr, 100 µl of [¹⁴C]BCB (25 mg/kg, 50 µCi/kg) in ethanol/propylene glycol, 3:7 (v/v) was applied to the shaved backs of the animals. Following topical application, rats were placed into Nalgene metabolism cages for the collection of urine and feces. Blood samples were collected at selected points (0, 15, 30, and 45 min, as well as 1, 3, 4, 6, 8, 12, 24, 48, and 72 hr). At the end of the 72-hr experiment, the animals were euthanized by carbon dioxide inhalation. The skin at the treatment site was washed with 50 ml of ethanol, and aliquots of the wash were analyzed for total radioactivity. Furthermore, the excised skin site was analyzed for radioactivity by tissue oxidation. Radioactivity adsorbed to the activated charcoal was desorbed by exhaustive extraction with ethyl acetate. The aluminum traps were washed in acetone (3 × 100 ml) and analyzed for total radioactivity.

Dispositional Studies. For each route of administration, rats were housed in glass metabolism cages, and exhaled radiolabel was collected for 72 hr. [¹⁴C]BCB equivalents associated with exhaled organics were trapped with 2-methoxyethyl ether (Mallinckrodt Chemical, Paris, KY), and ¹⁴CO₂ 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, 48, and 72 hr following administration of [¹⁴C]BCB. Urine (6, 12, 24, 48, and 72 hr) and feces (24, 48, and 72 hr) were also collected throughout the experiments. At the end of the 72-hr experiment, the animals were euthanized by carbon dioxide inhalation. Blood was collected immediately from the posterior vena cava into a heparinized syringe, and major tissues (liver, lungs, kidneys, heart, brain, stomach, spleen, testes, adipose, small and large intestine, as well as gastrointestinal contents) were excised, weighed, and stored at 80°C until analyzed (Winter and Sipes, 1993). Blood, feces, and tissue samples were analyzed for total radioactivity by sample oxidation (Winter et al., 1992). Body composition estimates of 11% for adipose tissue, 8% for blood, 50% for muscle, and 16% for skin were used (Birnbaum et al., 1980; Matthews and Anderson, 1975).

Stability and Reactivity of BCB. To define the stability of BCB in both whole blood and plasma, [¹⁴C]BCB (28 µg, 0.4 µCi) was incubated at room temperature or 37°C with whole blood and plasma (150 µl) for various times. At the end of the incubation, samples were extracted and analyzed by HPLC as described below. To determine the role of free sulfhydryl moieties, N-ethylmaleimide (40 mM) was added to the incubation before BCB. Furthermore, BCB was incubated for 10 min in physiological phosphate buffer containing 25 mM of glutathione with and without N-ethylmaleimide (40 mM).

Data Analysis. The blood concentration-time data following iv bolus dosing were analyzed by both compartmental and noncompartmental methods. Disposition parameter values best describing a linear multicompartmental model, and assuming first-order kinetics for all processes, were determined by nonlinear regression analysis as previously described (Sauer et al., 1997).

HPLC Analysis of BCB and its Metabolites. Blood samples were measured directly for total radioactivity by scintillation counting (2 × 25 µl of whole blood). BCB and its metabolites were extracted from 150 µl of blood with 300 µl of ethyl acetate. The samples were vortexed, centrifuged, and organic extracts removed. The extraction procedure was repeated two more times, the extracts were pooled, and 150 µl of water was added. Analytes were forced into the aqueous fraction by evaporation of ethyl acetate via vacuum centrifugation. The samples (100 µl) were injected onto a 250 × 4.6-mm Whatman Partisil ODS-2 (10 micron) column (Whatman, Hillsboro, OR), and metabolites were eluted with a mobile phase of water and acetonitrile both containing 0.1% acetic acid at a flow rate of 1 ml/min with a total run time of 80 min as previously described (Sauer et al., 1997). For analysis of urinary metabolites, urine samples were diluted 1:2 (v/v) with acetic acid to approximately pH 4 and centrifuged to remove any precipitate. Prepared urine samples (100 µl) were analyzed by HPLC as described above. Urine samples were also incubated with -glucuronidase (2000 units/ml) or sulfatase (100 units/ml) for at least 24 hr and then analyzed by HPLC.

Metabolite Identification Using MS and MS/MS Analysis. For metabolite isolation and identification, pooled urine samples from each route of administration were diluted 1:2 (v/v) with 0.5% acetic acid. Samples were 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 ion source after on-column separation of analytes utilizing the same HPLC system described above or by direct flow injection at 0.3 ml/min. Ions with m/z values corresponding to putative metabolites were subjected to collision-induced dissociation with argon gas, and the subsequent product ion signal masses were analyzed to produce a product ion mass spectrum.

	Results

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Intravenous Administration. No BCB was detected in the blood following iv administration (including the 1-min time point); however, 2-MGN was found in the blood. Although 2-MGN was found in relatively high levels (C_max 7.3 µg/ml), its concentration declined rapidly and by 1 hr was below the limit of detection. Within 6 hr, >40% of the dose of [¹⁴C]BCB had been excreted into the urine (fig. 1A). Recovery of total radioactivity (at 72 hr) in the urine, feces, and tissues is shown in table 1. Only a small percentage of the dose appeared in the feces (4.1%), whereas 6.6% of the dose was exhaled as ¹⁴CO₂. Tissue oxidation indicated that only 4.9% of the dose was retained in tissues 72 hr after treatment, but 12.5% of the dose was bound to the erythrocyte fraction of the blood. No single tissue contained a large amount of the radioactive dose, instead the dose seemed to be evenly distributed throughout the body.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Cumulative recovery of total radioactivity in the urine and feces following (A) intravenous (8 mg/kg, 120 µCi/kg), (B) oral (80 mg/kg, 100 µCi/kg), and (C) topical (25 mg/kg, 50 µCi/kg) administration of [14C]BCB in male Fischer 344 rats. Note the break in the vertical axis of graph C. Data expressed as mean percentage of dose ± SD (N = 3).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Whole blood concentration of intact 2-methyleneglutaronitrile following intravenous bolus administration of [14C]BCB (8 mg/kg, 120 µCi/kg) and [14C]2-MGN (3.3 mg/kg, 120 µCi/kg) to male JVC Fischer 344 rats. Data represent mean ± SD (N = 3). The lines represent the nonlinear regression fit of the data [C = 2.63e0.38t + 3.15e0.01t for 1,2-dibromo-2,4-dicyanobutane (dashed line); C = 6.79e0.60t + 3.66e0.02t for 2-methyleneglutaronitrile (solid line)].

Oral Administration. Following oral administration of [¹⁴C]BCB, 72% of the dose was excreted into the urine, and 9.7% was excreted into the feces (table 1). Cumulative excretion of radioactivity in the urine and feces is shown in fig. 1B. The recovery pattern was virtually identical to that following iv administration, except for a lag in the excretion of ¹⁴C equivalent into the urine and CO₂ likely due to absorption of BCB or its metabolites from the gastrointestinal tract. The radioactivity associated with the exhaled CO₂ was 7.5% of the dose. Although tissues retained 3.5% of the dose 72 hr after treatment, the dose was evenly distributed among tissues including the liver and gastrointestinal tract. Approximately 2.6% of the dose was bound to the blood at the end of the experiments. No parent BCB could be detected in the blood following oral exposure; however, a small amount of 2-MGN was present in the blood following oral administration (C_max 0.32 µg/ml). Although it seems that at least 85% of dose was absorbed following oral administration, it is unlikely that BCB is absorbed intact.

Topical Administration. Recovery and elimination of total radioactivity following topical exposure is shown in table 1. Approximately 83% of the radioactive dose was associated with the activated charcoal, skin trap, and skin at the site of application. Cumulative excretion of absorbed radioactivity is shown in fig. 1C. The excretion pattern of absorbed ¹⁴C equivalent was similar to that observed following iv or oral administration; 4.9% of the dose was recovered in urine, and 0.6% was recovered in feces. Little radioactivity was associated with the exhaled CO₂ (0.4%) and organic vapors (0%), whereas approximately 0.5% of the dose was bound to the skin at the site of application. Although blood contained only a small amount of radioactivity 72 hr after treatment (0.4% of the dose), approximately the same amount was retained by the tissues (0.5% of the dose). Thus, it seems that less than 12% of the dose was absorbed following topical application.

Stability and Binding of BCB in Blood. When incubated with whole blood, BCB was rapidly converted to 2-MGN. In less than 1 min, no BCB could be detected in the incubation mixture. Plasma also converted BCB to 2-MGN; however, the rate of conversion was significantly slower than whole blood. Following 10 min of incubation, ~5% of the spiked BCB remained intact. Interestingly, the formation of 2-MGN could be inhibited by conjugating free-sulfhydryls with N-ethylmaleimide. Glutathione-containing buffers were also found to readily catalyze the conversion of BCB to 2-MGN (data not shown). When added to buffers containing glutathione, BCB was readily converted to 2-MGN. Oxidized glutathione (glutathione disulfide) was also a product of this reaction. Similar to plasma and blood, glutathione-mediated conversion could be reduced with N-ethylmaleimide preincubation.

Determination of Blood Metabolites. Following iv and oral administration of [¹⁴C]BCB, HPLC analysis of blood revealed one major radioactive peak (R_T 24 min). A second peak was also observed at later times following iv administration (R_T 19 min). Representative GC-MS analysis of the isolated peaks is shown in fig. 3. The mass spectrum of the primary metabolite had a molecular ion corresponding at m/z 106. Furthermore, the EI spectra of this metabolite was characteristic of 2-MGN (fig. 4, panel II). The HPLC and GC retention time and mass spectra of this putative metabolite in the samples were compared with authentic standards to verify compound identity. The secondary peak (R_T 19 min) was not identified because of its low concentration in blood.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Representative spectra of major blood and urinary metabolites obtained from male Fischer 344 rats following administration of [14C]BCB. (A) Representative GC-MS EI spectra of 2-methyleneglutaronitrile (RT 24 min). (B) Representative LC-MS/MS (esi) product ion spectra of [M-H] m/z 268, N-acetyl-S-(2,4-dicyanobutane)-L-cysteine (RT 23 min). (C) Representative LC-MS/MS (esi) product ion spectra of [M-H] m/z 284, N-acetyl-S-(2,4-dicyanobutan-2-ol)-L-cysteine (RT 19 min). (D) Representative LC-MS/MS (esi) product ion spectra of [M+H] m/z 109, propanoic-1,3-dicyanide (RT 9 min).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Metabolic scheme proposed for 1,2-dibromo-2,4-dicyanobutane in the male Fischer 344 rat. 1,2-Dibromo-2,4-dicyanobutane (I), 2-methyleneglutaronitrile (II), N-acetyl-S-(2,4-dicyanobutane)-L-cysteine (III), N-acetyl-S-(2,4-dicyanobutan-2-ol)-L-cysteine (IV), and propanoic-1,3-dicyanide (V). *, location of 14C label; GS, glutathione conjugate; S-Cys-N-Acetyl, mercapturate conjugate; GSH, glutathione; R-SH, free sulfhydryl; GSSG, glutathione disulfide; RSSG, mixed disulfide.

Determination of Urinary Metabolites. Following reversed phase HPLC analysis of urine from rats treated intravenously, orally, and topically with [¹⁴C]BCB, several radioactive peaks were detected (fig. 5). Each route of administration showed similar metabolite profiles. A quantitative accounting of the urinary metabolites following each route is shown in table 2. Neither -glucuronidase nor sulfatase incubation had an effect on the metabolic profiles of the urine. The principle urinary metabolite (R_T 23 min) was identified as N-acetyl-S-(2,4-dicyanobutane)-L-cysteine (fig. 4, panel III). Another significant urinary metabolite (peak R_T 19 min) was identified as N-acetyl-S-(2,4-dicyanobutan-2-ol)-L-cysteine (panel IV). The metabolite peak R_T 9 min was identified as propanoic-1,3-dicyanide (panel V). Although no parent was detected in the urine, a minor amount (<0.1% of the dose) of 2-MGN was observed (panel II).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   HPLC radiochromatograms of pooled urine samples (6-, 12-, and 24-hr collection; 48 hr included for topical) from male Fischer 344 rats following (A) intravenous (8 mg/kg, 120 µCi/kg), (B) oral (80 mg/kg, 100 µCi/kg), and (C) topical (25 mg/kg, 50 µCi/kg) administration of [14C]BCB. The peaks are identified by letters that correspond to those shown on table 2.

	Discussion

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The work described here represents a comprehensive study of the absorption, disposition, metabolism, and excretion of BCB in the male Fischer 344 rat. Foremost among our observations was that no parent compound could be detected in the blood at any time regardless of the route of administration, including iv. The debrominated product of BCB, 2-MGN, was observed following both iv and oral administration. Although a portion of the dose was absorbed percutaneously, at no time was BCB or its metabolites detected in the blood. When administered topically, BCB was absorbed significantly slower and to a lesser degree than when given orally. This lack of detection in the blood is likely because of the slow absorption of BCB in conjunction with its rapid metabolism. Thus, regardless of the route of administration, it seems that this compound undergoes substantial presystemic clearance, and it is improbable that any BCB that is absorbed intact escapes debromination to gain access to the systemic circulation. Because following either oral or topical administration no BCB actually reaches the systemic circulation as intact compound, tissue exposure to BCB is exceedingly small or even nonexistent. However, tissue exposure to 2-MGN is substantially greater.

Immediately following iv BCB administration, 2-MGN was found in relatively high levels, and its concentration-time profile could adequately be described by a biexponential equation, consistent with a linear two-compartmental model. Interestingly, when 2-MGN itself was iv administered, its concentration-time profile was nearly identical to that observed following BCB administration. Thus, it seems that BCB is immediately converted to 2-MGN after iv administration. Following administration of 2-MGN, there was a relatively limited distributive phase, which correlated with a steady state volume of distribution (0.68 liters/kg) that is not especially large, suggesting that only a limited amount of 2-MGN distributes to the tissues. As a consequence of its low rate of clearance (11.5 ml/kg · min) and small apparent volume of distribution, 2-MGN has a disposition half-life of 60.6 min.

As discussed above, the principle metabolite of BCB detected in the blood was 2-MGN. The rapid formation of 2-MGN suggests that the bromine moieties of BCB are extremely labile and readily eliminated. Whole blood and, to a lesser extent, plasma possess the ability to debrominate BCB. This rapid conversion by whole blood is likely responsible for the inability of our assay to detect BCB following iv administration. The debromination of BCB seems to be mediated by a free-sulfhydryl-dependent biotransformation pathway. Incubation of BCB in glutathione-containing solutions results in the formation of 2-MGN, as well as oxidized glutathione (glutathione disulfide). From the in vitro incubation experiments, it seems that two glutathione molecules are required for the total debromination of BCB. However, these nucleophilic attacks do not result in the formation of a stable glutathione conjugate intermediate. An analogous reaction has been previously described by Livesey et al. (1982). This sulfhydryl-dependent mechanism involves the nucleophilic attack of glutathione on a dihaloalkyl substrate resulting in S-(beta-haloalkyl)glutathione formation; subsequent attack of a second thiol on the sulfur atom of the conjugate yields glutathione disulfide, halide ion, and an alkene product. It seems that the rapid conversion of BCB to 2-MGN follows this same mechanism. Interestingly, 2-MGN was not a major metabolite detected in the urine, and it seems that following formation the majority of 2-MGN is further metabolized, and its metabolites are then excreted into the urine.

No information is available in the literature on the biotransformation, toxicity, or carcinogenicity of 2-MGN. However, 2-MGN is structurally similar to several previously investigated nitrile-containing compounds and seems to follow common metabolic pathways (Fennell et al., 1991; Silver et al., 1982). Following formation, 2-MGN undergoes glutathione conjugation or oxidation, both of which are directed at the unsaturated portion of the molecule. The primary route of biotransformation for 2-MGN is glutathione conjugation, which results in N-acetyl-S-(2,4-dicyanobutane)-L-cysteine as the principal urinary metabolite (38-46% of the radiolabel in the urine). A secondary route of metabolism is oxidation, which apparently results in the formation of an epoxide intermediate (2-methyleneglutaronitrile oxide). This reactive intermediate can be conjugated by glutathione or hydrated, resulting in the formation of N-acetyl-S-(2,4-dicyanobutan-2-ol)-L-cysteine and 1,2-dihydroxy-2-methylglutaronitrile, respectively. Further oxidation of 1,2-dihydroxy-2-methylglutaronitrile leads to the loss of a methyl group (carbon 1 of the molecule) and the formation of propanoic-1,3-dicyanide (~9% of the dose). Loss of carbon 1 results in the exhalation of radiolabel in the form of ¹⁴CO₂ (~8% of the dose). Interestingly, the cyano moieties of BCB seem to avoid metabolism, and unlike other cyano-containing compounds, BCB dose not liberate cyanide. This difference is probably due to structural considerations in the molecule (Silver et al., 1982). A schematic representation for the proposed metabolic fate of BCB consistent with the data reported here is shown in fig. 4.

Another important finding was the significant amount of radiolabel covalently bound to the erythrocyte fraction of the blood. This bound fraction accounted for the presence of ¹⁴C equivalents 72 hr after BCB administration and could not be displaced with various solvent extractions. Although BCB is rapidly converted to 2-MGN, in vitro incubation of BCB with whole blood resulted in an increased amount of covalent binding with time. Furthermore, incubation of 2-MGN with whole blood also resulted in binding that increased with time. The kinetic profiles for binding were similar for BCB and 2-MGN. From these data, it seems that BCB itself is not responsible for covalent binding, but instead 2-MGN seems to mediate the majority of erythrocyte binding. Alkylation of free-sulfhydryls, which inhibits the conversion of BCB to 2-MGN in the blood, resulted in a significant decrease in the amount of binding for both BCB and 2-MGN. It is, however, unclear in the case of 2-MGN whether free-sulfhydryls are required for further biotransformation or as targets for binding. Thus, it seems that the conversion of BCB to 2-MGN as well as the presence of free-sulfhydryls in whole blood are required to achieve maximal covalent binding. Furthermore, the amount of in vivo erythrocyte binding was dictated by the route of administration. When BCB was administered orally, 2.6% of the dose was bound, whereas 12.5% was bound following iv administration. The difference in binding is likely due to the high first-pass hepatic metabolism (glutathione conjugation) of 2-MGN following oral administration. Thus, this metabolite is conjugated and unable to react with macromolecular binding sites on the erythrocyte.

A portion of the dose was also retained in the tissue (0.8-4.9%). Interestingly, the binding was not tissue specific and was evenly distributed among tissues. Such a distribution pattern is likely because of one of the metabolites of BCB binding to vascular tissue throughout the body or because of the presence of blood-bound ¹⁴C equivalents present in tissues. Interestingly, the route of administration had no effect on tissue binding of BCB, including binding to hepatic and gastrointestinal tissues. BCB has been previously shown to be a weak sensitizing agent in humans (Hausen, 1993; Tosti et al., 1995). This adverse effect may be related to the macromolecular binding of BCB and the formation of immunoreactive adducts (haptens). However, further investigation is required to determine if BCB binding to tissues plays a significant role in its ability to cause systemic sensitization.

In summary, results of this study indicate that BCB is absorbed to various degrees following oral and topical administration. Although absorption through the skin is significantly slower than from the gastrointestinal tract, in both cases it is unlikely that BCB is absorbed intact. However, any BCB that reaches the systemic circulation intact is converted to 2-MGN as a result of the extremely rapid debromination of BCB catalyzed by free-sulfhydryls in the blood. Thus, tissue exposure to BCB, regardless of the route of administration (including iv), is expected to be exceedingly low. However, this is not the case for its primary metabolite, 2-MGN, which is biotransformed and eliminated at a significantly slower rate. Furthermore, the debrominated metabolites of BCB seem to be responsible for the majority of macromolecular binding observed in the blood. Thus, any toxicity or carcinogenicity observed following BCB administration is likely mediated by 2-MGN or one of its primary metabolites (i.e. epoxide intermediate). Future studies should, therefore, determine the early tissue disposition of 2-MGN, as well as the organ(s) responsible for its metabolism. Furthermore, because a significant portion of the dose was found bound to tissues and the erythrocyte fraction of the blood, the possible toxic ramifications of these observations should be investigated.