Mode of Action and Pharmacokinetic Studies of 2-Butoxyethanol in the Mouse with an Emphasis on Forestomach Dosimetry

Torka S. Poet*,1, Jolen J. Soelberg*, Karl K. Weitz*, Terryl J. Mast*, Rodney A. Miller{dagger}, Brian D. Thrall* and Richard A. Corley*

* Battelle, Pacific Northwest Division, and {dagger} Battelle, Columbus Operations, P.O. Box 999, Richland, Washington 99352

Received August 23, 2002; accepted November 12, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic inhalation studies with 2-butoxyethanol (BE) conducted by the National Toxicology Program identified the forestomach and liver of B6C3F1 mice as target organs for tumorigenicity (NTP, 2000Go). Previous studies have shown that the liver tumors likely resulted from chronic hemolysis-induced oxidative stress. For the forestomach lesions seen in mice, chronic contact irritation (cytotoxicity) and regenerative hyperplasia are hypothesized to result in forestomach tumor development. To test this hypothesis, several experiments were conducted to address the sensitivity of the mouse forestomach to BE administered by various routes. Oral administration of undiluted BE was shown to cause irritation and a compensatory proliferative response in the mouse forestomach, confirming that direct contact between the forestomach and BE, which can occur via grooming of BE condensed on the fur during inhalation exposures, can cause irritation. However, only small amounts of BE (<10 mg/kg) were detected on the fur of mice at the end of 6-h, whole-body or nose-only inhalation exposures to the highest concentration used in the NTP chronic inhalation studies (250 ppm). Furthermore, no significant differences were detected in the end-exposure blood concentrations of BE and butoxyacetic acid (BAA) between these types of exposures. In addition, parenteral administration of BE (ip and sc injection) also resulted in forestomach lesions, indicating that there may be sources other than grooming for BE- or BAA-induced forestomach irritation. In the pharmacokinetic study, BE and, to a lesser extent, BAA was eliminated more slowly from the forestomach tissue of mice than from blood or other tissues, following either oral gavage or ip injection. The forestomach was the only tissue with detectable levels of BE at 24 h. BE and BAA were both excreted in the saliva and were present in stomach contents for a prolonged period of time following these routes of exposure, which may further contribute to forestomach tissue dosimetry. Thus, there appear to be multiple mechanisms behind the increased levels of BE and BAA in the forestomach tissue of mice, which together can contribute to a prolonged contact irritation, compensatory hyperplasia, and tumorigenicity in mice. The relevance of these effects in humans, who lack a forestomach, is questionable.

Key Words: glycol ethers; 2-butoxyethanol; butoxyacetic acid; forestomach, dosimetry.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2-Butoxyethanol (BE; ethylene glycol butyl ether; EGBE) is currently the largest production volume glycol ether, with over 650 million pounds produced annually in the U.S. and Europe (ACC, 2000Go). BE is a key ingredient in hundreds of products, including industrial and consumer hard surface cleaners and water- and solvent-based paints and coatings. Human health-risk assessments and exposure guidelines developed for 2-butoxyethanol have generally focused on hemolysis as the most sensitive toxic endpoint (ECETOC, 1994Go; EPA, 1999Go). Numerous studies have shown that humans are significantly less sensitive to the hemolytic effects of 2-butoxyethanol’s major metabolite, butoxyacetic acid (BAA), than are rats or mice (Ghanayem and Sullivan, 1993Go; Udden, 1994Go; Udden and Patton, 1994Go). However, in a recent chronic inhalation toxicity study conducted by the National Toxicology Program, the forestomach of female B6C3F1 and the liver of male B6C3F1 mice were also identified as potential target organs for tumorigenicity (NTP, 2000Go). Given these results, and the general lack of genotoxicity of BE (Elliot and Ashby, 1997Go), several studies have recently been conducted with the mouse, or are underway, to determine the relevance of the NTP findings for humans.

Hemolysis appears to be a root cause for the induction of liver tumors in male B6C3F1 mice (progression of hepatocellular adenomas to carcinomas and hemangiosarcomas). Chronic hemolysis and the accumulation of hemosiderin in the Kupffer cells results in oxidative stress in the livers of male mice, which are particularly sensitive due to an inherently low antioxidant capacity compared with female mice, rats, and humans (Kamendulis et al., 1999Go; Klaunig et al., 1998Go; Park et al., 2002Go). Vascular endothelial cells are also continually exposed to hemolysis byproducts, which may represent an additional stress on the hepatic vascular endothelial cells. Since hemolysis is unlikely to occur in humans (Ghanayem et al., 1987Go; Klaunig et al., 1998Go; Udden, 1994Go; Udden and Patton, 1994Go) and Park et al.(2002)Go have shown that neither BE nor BAA affect the liver cells directly, it is questionable whether hepatic tumor production will be a factor for humans.

Although chronic hemolysis may conceivably contribute to forestomach lesions and subsequent tumor production observed in the NTP (2000)Go study in mice (Table 1Go) via localized blockage of capillaries, followed by ischemia reperfusion injuries, the majority of the available evidence points towards a chronic irritation (cytotoxicity) compensatory hyperplasia mechanism caused by localized high concentrations of BE or its major metabolite, BAA. Many glycol ethers, including BE, are known to irritate mucous membranes (Boatman and Knaak, 2001Go). The major alkoxyacetic acid metabolite, BAA, is likely to cause similar effects. Thus, chronic contact irritation from either consumption of BE deposited on the fur as a result of grooming, dissolution of BE in the mucous layers of the respiratory tract followed by mucociliary clearance and oral consumption, partitioning from systemic blood circulation, or local forestomach metabolism of BE to BAA are likely candidates for further study.


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TABLE 1 Incidences of Forestomach Lesions in Mice Exposed by Inhalation to 2-Butoxyethanol for 13 Weeks or Two Years (NTP, 2000Go)
 
Prior to the NTP studies, only limited, nonspecific data were available on the tissue distribution of BE that would have indicated the forestomach could be a potential target tissue following inhalation exposures. Ghanayem et al.(1987)Go showed that 48 h after male F344 rats were dosed with 14C-BE by oral gavage, the forestomach retained up to 4–6 fold more total 14C than other tissues, including the liver and kidneys. While this result was intriguing, neither the chemical form of the 14C in forestomach tissue was identified nor were other routes of exposure evaluated to determine if the forestomach accumulation was strictly a portal-of-entry effect.

The tissue disposition of radio-labeled BE in mice was investigated in a series of autoradiographic studies by Green et al. (2001aGo,bGo; 2002)Go and Bennette, et al. (2001)Go. In these studies, high local concentrations of radioactivity were present in the buccal cavity, esophagus, and stomach tissues following either intravenous or short-term inhalation exposures. In the subchronic and chronic inhalation studies conducted by NTP (2000)Go, treatment-related lesions were limited to the forestomach; no lesions were noted in the esophagus or buccal cavity. Radioactivity was also detected on the fur of mice exposed via inhalation, supporting earlier conclusions from the NTP chronic inhalation study that oral consumption via grooming of the fur may be an important factor in forestomach dosimetry, chronic irritation, and subsequent tumor promotion. In support of this hypothesis, Green et al.(2001a)Go also demonstrated that forestomach lesions, similar to those seen in the subchronic inhalation study, could be produced in female B6C3F1 mice given single oral doses of BE at dose levels >=150 mg/kg. However, Green et al. (2001b)Go also detected 14C in mouse forestomachs following intravenous injection of 2-butoxyethanol, indicating that the mouse forestomach tissue may have a propensity for accumulating 14C, although the chemical form and the source(s) for the accumulation was not known.

The purpose of this study was, therefore, to investigate the pharmacokinetics of butoxyethanol and its major metabolite, butoxyacetic acid, in female B6C3F1 mice, with an emphasis on target tissue dosimetry and forestomach toxicity. This study was divided into five experiments as outlined in Table 2Go. The first experiment was to establish whether oral administration of undiluted, neat 2-butoxyethanol, to mimic grooming of BE deposited on the fur during inhalation exposures, is required to produce forestomach irritation and toxicity in male and female B6C3F1 mice or whether other routes of exposure can also result in comparable toxicity. The second experiment was to determine the target organ dosimetry and kinetics of BE and its major metabolite BAA in female mice, following oral and parenteral routes of exposure. The remaining three experiments were all designed to investigate several possible sources of BE and BAA in the forestomach and glandular stomach tissues of female mice, including deposition of BE on fur during inhalation studies and ingestion via grooming, the presence of BE and BAA in saliva, and the partitioning of BE and BAA in stomach tissues and stomach contents.


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TABLE 2 Study Outline
 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Spectroscopic grade BE (greater than 99% pure) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and BAA (greater than 99% pure) was obtained from Spectrum (San Francisco, CA). All other chemicals were reagent grade or better and were purchased from commercial sources.

Animals.
A number of experiments were designed to address target tissue dosimetry and potential forestomach lesions as outlined in Table 2Go. Adult male and female B6C3F1 mice used in the target tissue histopathology and fur deposition (inhalation) studies were purchased from Taconic Laboratory Animals and Services (Germantown, NY), the same source that was used in the NTP inhalation studies. For the inhalation exposures, slightly older (10-week-old) mice were used, in order to be of sufficient size to fit in the nose-only exposure system. Female B6C3F1 mice appeared to be slightly more sensitive to the induction of forestomach lesions than males in the subchronic and chronic NTP inhalation studies (Table 1Go; NTP, 2000Go). Therefore, adult female B6C3F1 mice approximately 8 weeks of age, purchased from Charles River Laboratories (Raleigh, NC), were used in the tissue dosimetry and pharmacokinetics studies.

The rooms in which the animals were housed were maintained on a 12-h light/dark cycle and designed to maintain adequate temperatures, relative humidity, and airflows. Deionized water (reverse osmosis) and Purina Certified Rodent Chow #5002 (Purina Mills, Inc., St. Louis, MO) were provided ad libitum. The animals were acclimated to the laboratory for at least 5 days prior to dosing or exposure. During the acclimation period, animals were uniquely marked with a tail tattoo, weighed, and randomly assigned to subgroups, based upon targeted sacrifice times.

Experiment 1: Target tissue histology.
Previous studies using drinking water as a route of administration failed to produce forestomach toxicity (NTP, 1993Go). This lack of forestomach irritation could have been the result of either a lower dose rate or a dilution of BE in water to concentrations that were not irritating to the mucosa. Therefore, no dosing vehicle was used in this study in order to determine if contact between the forestomach and undiluted (neat) BE, as would occur following grooming of BE condensed on the fur following whole body inhalation exposures, could cause forestomach lesions similar to those observed in the NTP studies.

Preliminary studies were conducted in groups of male and female mice, 5/sex/dose, at dose levels of 0 (saline control), 100, 400, or 800 mg/kg/day for 5 consecutive days, to determine if animals would tolerate the treatment regimen and to set the dose levels in the definitive study. Since no overt clinical signs were observed at 800 mg/kg/day, a few remaining animals (5/sex) were administered 1200 mg/kg/day for three consecutive days. Only these highest-dose animals demonstrated a significantly decreased hematocrit (~15% in males and 28% in females). Based upon results in the preliminary study, male and female B6C3F1 mice, up to 16/sex/dose, were treated by oral gavage at initial dose levels of 0 (saline control), 400, 800, or 1200 mg/kg/day of neat (undiluted) 2-butoxyethanol. Half the animals (8/sex/dose) were targeted for forestomach toxicity evaluations, with the remainder reserved for evaluations of liver effects. Feed was withheld from the mice for approximately 4 h prior to dosing and returned approximately 2 h after dosing, to simulate the brief fasting conditions associated with inhalation studies. After two days of dosing, mortality was unexpectedly observed at all dose levels. Therefore, dose levels for each group were arbitrarily reduced by half (i.e., to 200, 400, or 600 mg/kg/day) in an effort to improve survival. Dosing was discontinued when, after two additional doses (four doses total), mortality did not improve.

Each surviving animal was weighed and necropsied the day after the final dose. Stomach, liver, and spleen weights were recorded and blood was drawn by cardiac puncture for determination of the hematocrit. Slices of forestomach and glandular stomach tissues, as well as portions of each liver, were fixed in neutral buffered formalin for histopathological evaluations. Since the liver and hemolytic effects were used to design more definitive studies on the oxidative stress mechanisms (Park, et al., 2002Go), only the forestomach histopathology methods and results are presented.

Paraffin-embedded forestomach tissues were serially sectioned and stained with hematoxylin and eosin (H&E) or antibodies against proliferating cell nuclear antigen (PCNA). Histological evaluations of H&E sections were conducted to determine evidence of necrosis and/or irritation of the gastric mucosa. The primary determination of cells in S-phase was accomplished by analyses of PCNA incorporation, according to Eldridge and Goldsworthy (1996)Go.

To investigate if parenteral administration of BE could also produce forestomach lesions in mice, additional animals were administered saline solutions of BE by either intraperitoneal (ip) injection, which results in first-pass metabolism in the liver, or subcutaneous (sc) injection, which bypasses the first-pass liver metabolism. Dose levels were 0 (saline control) and 400 mg/kg/day for five consecutive days.

Experiment 2: Tissue dosimetry and pharmacokinetics.
Since forestomach irritation, a precursor to tumor formation, was induced following multiple routes of exposure (see Results), ip injection and oral gavage were chosen to facilitate accurate dosing and to compare oral vs. parenteral administration on forestomach tissue dosimetry. Groups of 30 female mice were administered target doses of 50 or 250 mg BE/kg BW by ip injection or 250 mg BE/kg BW by oral gavage. Each dose solution was prepared in physiologically buffered saline (pH 7.4) and analyzed for BE to confirm the target dose level. Each mouse was weighed and administered dose solutions at a rate of ~10 ml/kg by gavage or at a rate of 5 ml/kg body weight by ip injection. The dosing syringe was weighed before and after dosing to determine the actual volume delivered to each animal, and the dosing solutions were analyzed by gas chromatography/flame ionization detection to confirm the actual dosage.

At each scheduled sacrifice time (0.5, 1, 3, 6, 9, 12, and 24 h), animals were anesthetized in an 80% CO2 atmosphere and blood samples were collected by closed-chest cardiac puncture into Vacutainers® containing heparin, sealed and frozen on dry ice. The time of death was recorded at the completion of the blood draw and all animals were rapidly dissected to remove and weigh the kidney, liver, and stomach tissues. Stomach tissue was rinsed of its contents with ice-cold PBS, separated into forestomach (esophageal sphincter to limiting ridge) and glandular stomach (limiting ridge to pyloric sphincter) sections (each maintained as full thickness), and weighed. The tissues were flash-frozen in 4-ml amber vials and stored along with the blood samples at -80°C until analysis. The mice scheduled for sacrifice after 24 h were individually housed in glass metabolism cages, and urine was collected on dry ice from the 0–12 and 12–24-h intervals. These animals had food and water ad libitum during this time. The cages were rinsed with a minimal amount of deionized water (~20 ml) after the collection of each urine sample, and the cage wash sample was frozen until analysis. Blood, tissues, and urine were analyzed for BE and BAA by gas chromatography/flame ionization detection (GC/FID) or gas chromatography/mass spectrometry (GC/MS). Urine and cage wash samples were also acid-hydrolyzed to quantify potential conjugates of BE and BAA.

The areas under the curve (AUC) for both BE and BAA were calculated by the trapezoidal rule from 0–24 h (AUC0–24) and extrapolated to infinity (AUC{infty}) by addition of the quantity equal to the last measurable concentration divided by the elimination rate constant (Kel). The apparent first-order elimination rate constant (Kel) was calculated from a log-linear plot of the last three (or more) nonzero concentrations in the blood-concentration vs. time curve. The terminal phase blood half-life was obtained by dividing 0.693 by the elimination rate constant (Kel).

Experiment 3: Fur deposition following inhalation exposure.
Female B6C3F1 mice were exposed, either by whole-body or nose-only inhalation, to a single 6-h exposure to a target-concentration of 250 ppm BE, the highest concentration used in the NTP inhalation bioassay (2000)Go. BE vapor was generated on a heated (~105°C) glass surface and a metered air stream flowed through the generator (~20 l/min) and into the exposure chamber (whole-body or nose-only). BE chamber concentrations reached 90% of the stable final concentration within 10 min for the whole-body chamber and 3 min for nose-only chambers. Exposure duration was 6 h to the time 90% of the final concentrations were reached.

Whole body exposures were conducted in an 85-liter polycarbonate chamber. Mouse cages were suspended in the center of the chamber above a stainless-steel catch pan. BE vapor was introduced from multiple ports along the top and face of the chamber and was exhausted from ports below the catch pan. The generator airflow (~20 l/min) resulted in ~14 air changes per h. The distribution of BE vapor in the chamber was determined prior to the start of the study by comparing the concentrations from 3 different chamber positions.

The nose-only exposure unit was a flow-past design consisting of two concentric stainless-steel manifolds; one supplied fresh vapor to the breathing zone of each animal and one removed the expired and excess vapor. Airflow through the unit was ~20 l/min from the generator (inlet) with an exhaust flow of ~18 l/min, to prevent the animals from rebreathing their expired air. For concentration determination, three of the ports (one from each tier) were used to collect samples of the breathing atmosphere; samples were also collected from the inlet manifold using an additional port from the lowest tier. Uniformity of the test-article concentration in the exposure unit was determined prior to the study.

The exposure concentrations of BE were determined by gas chromatographic analysis of atmosphere samples collected on charcoal sampling tubes (ORBO-101) during the exposure. Known volumes of the chamber atmosphere were collected using a calibrated critical-orifice-controlled sampler at a constant flow rate of ~0.2 l/min for 3 min each. Toluene containing 1-phenylhexane internal standard was added to the primary charcoal bed after sample collection, to desorb the test chemical from the charcoal. The sample extracts were analyzed using an HP-5890 gas chromatograph (GC), as described below. Nominal exposure concentrations were calculated using the mass of test article pumped, the total time during which the metering pump was running, and the dilution airflow into the chamber. This value was compared to the average exposure concentrations obtained by the charcoal sampling tubes.

At the end of each exposure (nose-only and whole-body), five mice were sacrificed using CO2, transferred to individual beakers, and washed 2x in 50 ml of hot water. Each fur rinse was stored frozen (-80°C) until analyzed. This fur washing procedure was similar to that used by Tyl, et al.(1995)Go to determine the deposition of ethylene glycol on the fur of rats and mice exposed to ethylene glycol aerosols.

An additional 5 mice/exposure were anesthetized immediately after inhalation exposures, using 70% CO2, and samples of blood were collected via the retro-orbital sinus. Each blood sample was stored frozen (-80°C) until analysis. The final groups of 5 mice/exposure were placed in individual metabolism cages (Lab Products, Seaford, Delaware) for an 18-h urine collection. The urine samples were collected over dry ice. Following urine sample collection, each metabolism cage was rinsed with ~25 ml of distilled water. Each urine sample and cage rinse was stored frozen (-80°C) until analysis.

Experiment 4: Excretion into saliva.
To determine the kinetics of BE and BAA in blood vs. saliva, groups of mice were administered a target dose of 250 mg BE/kg body weight (bw), either by ip injection or oral gavage. Salivation was induced by injecting the cholinergic agonist, pilocarpine, a few min before beginning to collect saliva. Groups of mice (4 animals/dose/time) were anesthetized with an ip injection of ketamine:xylazine (87:13; 100 mg/kg) at specified times up to 2.5 h postdosing. Five min after the animal was anesthetized, pilocarpine (1 mg/kg) was injected and a glass capillary tube was used to collect saliva from the oral cavity for up to 45 min. Aliquots of saliva were collected every 15 to 30 min (~100–200 µl). Blood was collected from the retro-orbital sinus (~100 µl) at the midpoint of each saliva collection interval and by cardiac puncture at the end of saliva collection.

Experiment 5: Retention in stomach contents.
To determine if stomach contents could act as a sink for BE or BAA and thereby provide a prolonged exposure to stomach tissues, an additional group of animals was dosed with a target of 250 mg BE/kg bw by ip injection. The same methods of dosing and tissue collection used for the tissue dosimetry and pharmacokinetics studies (Experiment 2) were used for this additional group of animals, and blood, BE, and BAA were quantified in stomach tissue and stomach contents. The sacrifice times for this group were 3, 6, and 9 h and were chosen based on the results from the previous saliva study. In particular, forestomach tissue concentrations of BE were found to be significantly higher than other tissues by 6 to 9 h after dosing.

Analytical methods.
Blood, liver, forestomach tissue, glandular stomach tissue, cage washes, fur washes, urine, and saliva were all analyzed by acidification of a weighed quantity of sample (~100 mg each) with 0.9 ml of 0.9 M H2SO4. Approximately 0.25g of Na2SO4 (to improve extraction efficiencies) was added and the samples were extracted with an equal volume of ethyl acetate containing known concentrations of the internal standards: ethoxyethanol and ethoxyacetic acid. H2SO4, and ethyl acetate volumes were adjusted based on sample weights.

To determine the extent of conjugation of BE and BAA, aliquots (~100 µl) of urine and cage wash samples were hydrolyzed by the addition of HCl (~200 µl 6.0 N HCl) and incubated for 1 h at 110°C. Following incubation, 0.25g Na2SO4 was added to each sample and extracted with 200 µl ethyl acetate containing known concentrations of ethoxyethanol and ethoxyacetic acid internal standards.

Quantitation of BE and BAA in blood, glandular stomach tissue, cage wash, urine, fur washes, and saliva were performed using a Hewlett Packard 6890 Gas Chromatograph with flame ionization detection (Hewlett Packard, Avondale, PA). Separation and quantification were achieved with a 30 m x 0.32 mmid x 1.0 µm-film thickness Stabilwax DA capillary column (Restek, Bellefonte, PA). Splitless injections of 2.0 µl of extract were conducted at an injector temperature of 210°C. The initial oven temperature was 80°C for 1 min, increased at 20°C/min to 240°C, and held for 1 min. Hydrogen was used as the carrier gas at 25 psi for 5 min, then rapidly increased to 30 psi. The FID temperature was 275°C. Under these conditions, ethoxyethanol, butoxyethanol, ethoxyacetic acid, and butoxyacetic acid had retention times of ~2.4, 3.8, 6.9, and 7.9 min, respectively. Slight variations in retentions times were observed for each matrix.

Due to the presence of interferences by GC/FID, quantitation of BE and BAA in liver, forestomach tissue, and stomach contents was performed on a Hewlett Packard 6890 gas chromatograph equipped with a Hewlett Packard 5973 Mass Selective Detector. Sample injections, columns, and oven conditions were similar to those described for GC/FID analysis. For GC/MS analysis, helium was used as the carrier gas, rather than hydrogen, at an initial pressure of 25 psi for 5 min, and rapidly increased to 30 psi. Quantification of butoxyethanol and butoxyacetic acid in liver was achieved by selected ion monitoring of m/z 31, 57, or 73 for BE at ~3.8 and of m/z 31 or 58 for BAA at 7.9 min retention time. The ions monitored for any given matrices varied due to detection limits, linearity of response, and background interferences. The appropriate ions were determined using matrix standards.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1: Forestomach histology.
Mortality, sometimes high (38–75%), was observed across treatment groups by the second day (Table 3Go). Although significant hemolysis was expected at the higher-dose levels, the mortality observed even at the lower-dose levels was not expected, based upon preliminary probe studies conducted at similar dose levels. The mortality was attributed to the inability of many of the mice to tolerate the specific dosing regimen (bolus dosing with neat BE while feed was withheld for 6 h). The early mortality resulted in a decision to reduce the dose levels across treatment groups to improve survival. Although the dosing regimen did not proceed as planned (e.g., animals were to be evaluated after 1 and 2 weeks of dosing for 5 days/week), and dosing was discontinued after 4 total doses/animal, the dosing did achieve the primary objective of the study: namely, to establish whether or not direct dosing with neat, undiluted BE can cause forestomach lesions.


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TABLE 3 Body Weights, Mortality, and Hematocrits from 16 Mice/Sex/Dose
 
Evaluation of H&E-stained slides from mice that tolerated the dosing with neat BE for four days resulted in dose-related forestomach lesions in all male and female mice that were similar to those reported in the NTP inhalation studies (NTP, 2000Go). These lesions included epithelial hyperplasia and inflammation of the forestomach. In the lower-dose groups (400/200 mg/kg/day), minimal to mild forestomach epithelial hyperplasia was observed in both male and female mice. Focal areas of increased layers of epithelial cells characterized these changes. In mice from the mid-exposure groups (800/400 mg/kg/day), minimal to moderate forestomach epithelial hyperplasia was observed in both genders. The epithelial hyperplasia, although appearing somewhat focal, was more expansive than observed in low-dose mice, and the epithelial thickening due to increased epithelial cell layers was more prominent. There were down-growths of proliferating epithelial cells into the lamina propria. In the mid-dose exposure group, several mice had significant infiltrates of neutrophils and mononuclear inflammatory cells in the submucosa and muscularis. Both genders of mice from the high-exposure groups (1200/600 mg/kg/day) had minimal to marked forestomach epithelial hyperplasia. Inflammation of the submucosa and muscularis was, again, commonly associated with the hyperplasia. A very tiny ulcer with minimal forestomach epithelial hyperplasia at its border was observed in one female control and minimal focal necrosis and forestomach epithelial hyperplasia at the limiting ridge (junction of forestomach and glandular stomach) was observed in another female control. The lesions in the control animals may have resulted from the gavage regimen (saline administered to controls) itself or were spontaneous changes of unknown etiology that are occasionally observed in mice (see Table 1Go).

Initial efforts to obtain a unit-length labeling index (ULLI) by counting the number of cells in S-phase per mm of the mucosa within the forestomach in at least four 0.25 mm random sections per animal proved inappropriate, due to the focal nature of the lesions. Therefore, analyses of PCNA labeling was limited to a qualitative assessment of the relative amount of S-phase cells in histologically normal forestomach epithelium versus the epithelium surrounding histological lesions. There was evidence of an increase in PCNA staining intensity in regions surrounding focal areas of hyperplasia and inflammation, as compared to either the forestomach epithelium of control mice or to histologically normal regions within the forestomach of BE-exposed mice. Lesions with focal regions of inflammation and thickening of the epithelium showed greater overall PCNA staining as well as clearer evidence of S-phase cells.

Minimal lesions, similar to those produced in the lower-dose (200–400 mg/kg/day) oral gavage groups, were observed in 1/6 of the ip-dosed mice and 2/6 of the sc-dosed mice following 4 days of dosing with 400 mg/kg/day. No lesions were observed in the controls. Representative pictures of forestomach lesions following each route of exposure are shown in Figure 1Go.



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FIG. 1. Representative H&E-stained forestomach sections (original magnification x200) from female B6C3F1 mice exposed to (a) PBS by oral gavage; (b) neat 2-butoxyethanol by oral gavage of 600 mg/kg/day; (c) PBS by subcutaneous injection; (d) 2-butoxyethanol by subcutaneous injection of 600 mg/kg/day; (e) PBS by intraperitoneal injection or (f) 2-butoxyethanol by intraperitoneal injection of 600 mg/kg/day.

 
Experiment 2: Tissue dosimetry and pharmacokinetics.
The actual doses (mean ± SD) of BE for the ip injections were 53.2 ± 2.8 and 261.4 ± 34.1 mg/kg. Actual doses for oral gavage averaged 265.2 ± 13.4. Regardless of dose or route of exposure, BE rapidly disappeared from blood and was no longer detectable after 1 h (Fig. 2Go). Following target doses of 250 mg/kg via either ip injection or gavage, BAA was detectable in the blood for up to 12 h (Fig. 3Go). The highest measured concentrations of BAA in blood and tissues were in the first samples, obtained 0.5 h postdosing. BAA concentrations at 0.5 and 1 h postdosing were approximately 5-fold lower in animals dosed with 50-mg/kg ip compared with the 250-mg/kg dose. No BAA was detected in blood from animals dosed with 50-mg/kg ip after 1 h.



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FIG. 2. Blood and tissue levels of BE following 50 or 250 mg/kg ip or 250 mg/kg oral doses from the dosimetry experiment. Symbols are mean ± SD of n = 4 animals for: X, liver; circle, forestomach; triangle, glandular stomach; square, kidney; diamond, blood.

 


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FIG. 3. Blood and tissue levels of BAA following 50 or 250 mg/kg ip or 250 mg/kg oral doses from the dosimetry experiment. Symbols are mean ± SD of n = 4 animals for: X, liver; circle, forestomach; triangle, glandular stomach; square, kidney; and diamond, blood.

 
With the exception of stomach tissue, BE levels in tissues paralleled the levels in blood, regardless of dose or exposure route. For the 250-mg/kg target doses to either route, forestomach and glandular stomach tissue concentrations were considerably higher than in blood or any other tissue measured. Levels of BE in the forestomach tissue persisted longer than in any other tissue (Fig. 2Go). For up to 3 h following ip dosing, BAA levels were highest in blood, kidney, and liver. After 3 h, BAA forestomach tissue concentrations were higher than for other tissues. Unlike in the forestomach, BAA concentrations in the glandular stomach tissue were similar to other tissues (Fig. 3Go).

Elimination half lives (T1/2) and areas under the curve (AUC) were determined for both BE and BAA (Tables 4Go and 5Go). For BE, both T1/2 and AUC were higher for gavage dosing than ip dosing. Regardless of route, the T1/2 and AUC were higher in forestomach than any other tissue. Blood T1/2 and AUC for BE could not be determined from data in the pharmacokinetic experiment since T1/2 was shorter than the earliest time points collected. The T1/2 for BAA in blood ranged from 1 h following ip injection to 2.1 h following a gavage dose.


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TABLE 4 BE Half-Lives and AUC in Blood, Saliva, and Tissues
 

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TABLE 5 BAA Half-Lives and AUC in Blood, Saliva, and Tissues
 
By 24 h, 53.8 and 48.4% of the total doses were eliminated in the urine as BE, BAA, or a conjugate following 250 mg/kg doses by ip and oral gavage, respectively (Table 6Go). The metabolite, BAA, represented the majority of the eliminated dose (50.8 and 37.5% for the ip and oral gavage, respectively). Very little unconjugated BE (<0.2% of dose) was detected in urine. Following acid hydrolysis, up to 3.3% of the administered dose was associated with a conjugate of BE, presumably a glucuronide. A conjugate of BAA was also detected, following acid hydrolysis, that represented 0–7.4% of the total dose, depending on the route and dose level. By comparison, acid hydrolysis of rat urine indicated that a conjugate of BAA is not eliminated in the urine of rats (Corley, et al., 1994Go).


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TABLE 6 Urinary Excretion of BE and BAA Following Intraperitoneal (ip) Injection and Oral Administration of 2-Butoxyethanol to Female B6C3F1 Mice
 
Experiment 3: Fur deposition following inhalation exposure.
An average of 205 ± 69 µg of BE was detected on the fur of five female mice exposed whole-body to 229 ppm BE vapor. Slightly less BE, 170 ± 52 µg/mouse, was detected on the fur of female mice exposed nose-only to 242 ppm BE. Residual BE on the fur of the nose–only-exposed mice was attributed to the use of a positive-pressure, flow-past exposure system. Most of the residual BE (~86%) was detected in the first 50-ml wash; thus, the method used to wash the fur was considered to be effective. Some evaporative losses of BE from the fur could have occurred during the time it took for the chamber to be shut down and evacuated and the animals sacrificed, prior to immersion in 50 ml of hot water, and some of the condensed material could have been consumed via grooming during the whole-body, but not nose-only, exposures. When corrected for the actual BE-exposure concentration, whole body exposures averaged 25% more BE on the fur than the corresponding nose-only exposures. While there was clear evidence that residual BE adsorbs to the fur of mice, the total dose measured postexposure that could be available to grooming and ultimately come into contact with the forestomach tissue averaged less than 10 mg/kg.

The concentration of BE in the blood of five female mice at the end of each exposure averaged 3.0 and 3.9 mg/l for whole-body and nose-only exposures, respectively. The concentrations of the major metabolite, BAA, in blood were 235 and 390 mg/l for the whole-body and nose-only exposures, respectively. These concentrations of BE and BAA were similar to the levels reported in the NTP chronic toxicokinetic study for equivalent exposure concentrations (Dill et al., 1998Go).

Low levels of unmetabolized BE (67.6–68.7 µg) were found in the urine of mice collected 0–18 h after both whole-body and nose-only exposures. The BE in urine was presumed to have come from the fur of the mice, since very little BE is expected to be excreted in urine unconjugated, based upon results from the Tissue Dosimetry and Pharmacokinetics study (Experiment 2) discussed above. Comparable amounts of free BAA were found in the urine following nose-only (2021 ± 537 µg) vs. whole-body (1783 ± 645 µg) exposure methods and were similar to levels reported in an NTP chronic toxicokinetic study (Dill et al., 1998Go).

Experiment 4: Excretion into saliva.
The actual doses for the salivary clearance studies were 258.0 ± 65.6 and 265.4 ± 41.8 mg/kg for the ip or oral gavage doses, respectively. Since the results from the tissue dosimetry and pharmacokinetics experiment (Experiment 2) indicated rapid blood-elimination kinetics and saliva and blood collections were carried out from 7 min to 2 h following ip injection and for up to 3 h following oral gavage. Peak blood and saliva BE concentrations were detected at the first time point (~15 min for blood, ~7.5 min for saliva) post-BE injection, regardless of route. Concentrations of BE in blood and saliva were nearly identical at all times and were below the level of detection after 1.3 h (Figs. 4aGo and 5aGo). BAA levels, however, did increase over the first 15–30 min and declined thereafter. Saliva BAA levels mirrored blood levels at all time points, albeit at approximately 4-fold lower levels (Figs. 4bGo and 5bGo).



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FIG. 4. BE and BAA levels in blood (squares) and saliva (circles) following an ip injection with 250 mg BE/kg. The times of saliva collection were at the midpoint of the 15-min collection interval. Symbols are mean ± SD of n = 4 animals.

 


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FIG. 5. BE and BAA levels in blood (circles) and saliva (squares) following oral gavage with 250 mg BE/kg. The times of saliva collection were at the midpoint of the 15-min collection interval. Symbols are mean ± SD of n = 4 animals.

 
Blood and saliva AUCs and T1/2s were determined from these exposures (Table 4Go), recognizing that these animals were under anesthesia and the effects of a cholinergic agonist (pilocarpine). The AUC and T1/2 for BE in blood and saliva were similar. The elimination T1/2 for BE in blood was about 10 min following an ip dose and 20 min following gavage dosing. Both the AUCs and T1/2s in blood and saliva were higher for BAA than BE. The T1/2 values for BAA were approximately 2-fold higher in blood than saliva. The blood elimination T1/2 for BAA in these studies was 1.4 and 1.6 h for ip and gavage dosing, respectively. In a study by Dill et al. (1998)Go, the T1/2 of elimination for BE and BAA, following inhalation dosing, were 3–4 and 22–31 min, respectively. In this study, the animals were anesthetized and given pilocarpine, so a direct comparison of half-lives is not possible. The higher AUC for BE in blood, following an ip dose, over the gavage dosing may be due to the lack of later time points from this study.

Experiment 5: Retention in stomach contents.
The actual dose administered, via ip injection, for the stomach contents experiment was 193.3 ± 23.1 mg/kg. Figures 6Go and 7Go show the stomach-content levels of BE and BAA at 3, 6, and 9 h post-ip dosing. The concentration of BE was higher in the stomach contents than in the forestomach tissue; no BE was detected in either blood or glandular stomach tissue at any time point in these animals, although it was present in the tissue dosimetry and pharmacokinetics experiment (Experiment 2) at a 26% higher dose.



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FIG. 6. Stomach and stomach-content concentrations vs. amounts of BE. Note: BE was below detection in the glandular stomach and blood at these time points following dosing with of 193.3 ± 23.1 mg/kg ip. Bars represent the mean ± SD of n = 4 animals.

 


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FIG. 7. Forestomach, glandular stomach, blood, and stomach-content concentrations and amounts of BAA following dosing with 193.3 ± 23.1 mg/kg ip. Bars are the mean ± SD of n = 4 animals.

 
The weight of the stomach contents was much greater than that of the forestomach tissue; therefore, on a total amount basis, BE levels were much greater in the stomach contents than in the tissue (Fig. 6Go). By comparison, total amounts of BAA were similar in stomach contents and forestomach and glandular stomach tissues at 3 and 6 h; by 9 h, BAA was only detected in the stomach contents (Fig. 7Go). Although only 3 time points were included in this experiment, the T1/2s and AUCs could be estimated for stomach contents and forestomach tissues (Table 4Go). The T1/2 for BE in the contents of the stomach was 4.8 h following ip injections, longest of all the tissues.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Following chronic inhalation exposures, female mice had increased incidences of combined forestomach squamous cell papillomas (benign) and carcinoma (Table 1Go; NTP, 2000Go). Given the lack of genotoxicity of BE (Elliot and Ashby, 1997Go), the forestomach tumors observed in these studies likely resulted from indirect mechanisms primarily involving chronic tissue injury. Since no studies have previously been conducted relating the dosimetry of BE and its major metabolite, BAA, with tissue injury (liver or forestomach), the pharmacokinetics of 2-butoxyethanol in female B6C3F1 mice was evaluated in this study with an emphasis on potential sources for forestomach tissue exposure to BE and BAA.

Oral dosing of neat BE to male and female mice resulted in dose-response irritation to the forestomach that was similar to the lesions observed in the NTP subchronic and chronic inhalation studies (Fig. 1Go). Forestomach lesions consisted of focal areas of irritation and epithelial hyperplasia. The proliferative responses were combined with increased PCNA immunohistochemistry of epithelial cells surrounding the lesions vs. normal cells in the forestomach. This is in contrast to earlier 2-week and 13-week drinking water studies conducted by the NTP (NTP, 1993Go), where no forestomach irritation was observed even at dose levels as high as 1400 mg/kg/day. These results indicate that high dose rates or at least locally high concentrations of BE or BAA may be required to produce forestomach irritation and toxicity.

Forestomach lesions were also observed in female mice following ip and sc injections of 400 mg/kg/day for 4 days, indicating that oral dosing is not required to produce toxicity (Fig. 1Go). These data suggest that systemic delivery of BE or its metabolites may cause forestomach lesions in the mouse similar to those observed in the NTP chronic inhalation bioassay. Thus, oral consumption via grooming of 2-butoxyethanol condensed on the fur of mice may not be the only explanation for forestomach irritation in the inhalation studies. Since forestomach irritation, a precursor to tumor formation, was induced following multiple routes of exposure, ip injection and oral gavage were chosen to compare oral vs. parenteral administration on forestomach tissue dosimetry.

The kinetics of BE in female mice showed a rapid conversion to BAA, with peak BE concentrations having occurred prior to the initial blood samples collected after 15 min (from the saliva study). Concentrations of BAA, as measured in the saliva study, indicated peak blood levels occurred at approximately 30 min postdosing. Analysis of tissue levels of BE and BAA indicated that both chemicals are retained in the stomach tissues, especially the forestomach region, to a greater extent than any other tissue. After oral gavage, initial BE levels in the mouse stomachs were much greater than any other tissue. These levels remained elevated for up to 12 h postdosing. Likewise, BAA levels in the forestomach following oral gavage did not parallel the other tissues and appeared to decline more slowly.

While stomach tissue concentrations of BE and BAA are expected to be elevated following oral gavage, remarkably similar trends in stomach tissue concentrations were observed after ip dosing. By 1 h postdosing, BE levels in the forestomach were greater than for any other tissue. This higher level of BE in the stomach (both forestomach and glandular stomach) was maintained throughout the 24-h tissue-sampling period. Following the 50 mg/kg ip dose, BE levels were still detected in the forestomach after 24 h, but were nondetectable in any other tissue, including blood, after 30 min. Forestomach BAA concentrations paralleled other tissues for up to 3 to 6 h, then showed an apparent decline in the elimination rate. It is possible that the relative increase in BAA in stomach tissue was a consequence of the retention or secretion of BE into the stomach followed by metabolism to BAA. A comparison of the alcohol and aldehyde dehydrogenase-mediated metabolism of BE in the mouse and rat indicates that forestomach tissues from the mouse may metabolize BE to BAA at a faster rate than in the rat (Green et al. 2001aGo).

Several studies, including those described herein, have indicated that the forestomach BE deposition is derived from a combination of several means: (1) grooming; (2) swallowing of mucous containing material; (3) salivary excretions and re-ingestion; and (4) grooming of BE present on the fur. High local concentrations of radioactivity were detected in the buccal cavity, salivary glands, esophagus, and stomach tissue following either intravenous (Bennette et al., 2001Go) or short-term inhalation (Green et al., 2001aGo,bGo) exposures to 14C-BE. While the amounts of BE recovered from the fur of mice exposed by whole-body or nose-only inhalation exposures were minimal in the present study (Experiment 3), the specific disposition of BE and BAA in the forestomach of female B6C3F1 mice following ip or oral gavage dosing (Experiment 2) supports these various methods for delivery of BE to the forestomach tissues.

The mouse stomach is anatomically very different from the human stomach. Unlike the human, the mouse stomach has two different regions. Food is initially deposited from the centrally located esophagus into the forestomach before it moves to the glandular stomach. The forestomach is a site of bacterial digestion with no counterpart in the human stomach. The mouse glandular stomach, like the human stomach, is secretory.

The design of the rodent forestomach allows for a slower rate of emptying of food from the stomach and enhanced digestion (DeSesso and Jacobsen, 2001Go). Both BE and BAA were present in the stomach contents at greater concentrations and total amounts than in the stomach tissues themselves (Figs. 6Go and 7Go). It is likely that small amounts of food remain in the forestomach and act as a sink for BE, which is swallowed or partitioned across the stomach mucosa. This sink, in turn, may act as a continual source for BE to forestomach tissues, which may further metabolize BE to BAA and maintain a localized tissue concentration long after BE and BAA have been cleared from the rest of the body (Fig. 8Go). In fact, locally high BE and BAA concentrations could have occurred in the tissues if these materials were associated with the epithelial cells and not distributed throughout the full thickness of the forestomach and glandular stomach as they were collected and analyzed. It is unlikely that BE and BAA preferentially bind to stomach tissue macromolecules, since the partition coefficients for BE and BAA are not significantly different in the stomach vs. any other tissue of rats or mice (Corley et al., 1998; Farris, 1998Go; Lee et al., 1998Go). A PBPK model for BE is currently being modified to include the forestomach and to further investigate the potential mechanisms leading to the forestomach disposition of BE and its metabolite, BAA.



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FIG. 8. Potential sources of BE and BAA in stomach tissues following parenteral injections, atmospheric exposures, or dermal exposures.

 
The tissue-specific dosimetry is important in relation to the forestomach lesions since they are most likely the result of prolonged exposure-induced irritation. The ip and oral gavage studies reported here demonstrate that the elimination of BE is slower from the forestomach than the blood and other well-perfused tissues such as liver and kidney, and BE and BAA are excreted in the saliva. This is in agreement with the iv, oral, and inhalation radiotracer studies reported by Green et al. (2001aGo,b)Go and Bennette et al.(2001)Go. Since high levels of BE and BAA were present in the stomachs of mice following several routes of exposure, it is likely that the chemical in this tissue comes from multiple sources, including grooming of fur containing BE, systemic blood circulation of BE and BAA, ingestion of BE and BAA from the buccal cavity (salivary excretion and clearance of mucus from the respiratory tract), and possibly repartitioning from the stomach contents (Fig. 8Go). However, it should be kept in mind that humans have no organ that is either morphologically or functionally comparable to the mouse forestomach. The notable differences between rodent and human stomach function and morphology are significant when considering the implications of mouse forestomach tumors to human risk assessment. In addition, although prolonged contact to an irritant can lead to damage and regenerative hyperplasia, a layer of mucus protects the glandular stomach.


    ACKNOWLEDGMENTS
 
We thank T. Curry, Kristine Studniski, and Theresa Luders for their exceptional help in animal dosing, sample collection, and handling. This research was supported by the Ethylene Glycol Ethers Panel of the American Chemistry Council.


    NOTES
 
Presented in part at the 41st Annual Meeting of the Society of Toxicology, March 17–21, 2002, Nashville, TN.

1 To whom correspondence should be addressed at Chemical Dosimetry, Battelle, Pacific Northwest Division, P.O. Box 999, MSIN P7-59, Richland, WA 99352. Fax: (509) 376-9064. E-mail: torka.poet{at}pnl.gov. Back


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