Determination of age and gender differences in biochemical processes affecting the disposition of 2-butoxyethanol and its metabolites in mice and rats to improve PBPK modeling.

2-Butoxyethanol (BE) is the most widely used glycol ether solvent. BEs major metabolite, butoxyacetic acid (BAA), causes hemolysis with significant species differences in sensitivity. Several PBPK models have been developed over the past two decades to describe the disposition of BE and BAA in male rats and humans to refine health risk assessments. More recent efforts by Lee et al. [Lee, K.M., Dill, J.A., Chou, B.J., Roycroft, J.H., 1998. Physiologically based pharmacokinetic model for chronic inhalation of 2-butoxyethanol. Toxicol. Appl. Pharmacol. 153, 211-226] to describe the kinetics of BE and BAA in the National Toxicology Program (NTP) chronic inhalation studies required the use of several assumptions to extrapolate model parameters from earlier PBPK models developed for young male rats to include female F344 and both sexes of B6C3F1 mice and the effects of aging. To replace these assumptions, studies were conducted to determine the impact of age, gender and species on the metabolism of BE, and the tissue partitioning, renal acid transport and plasma protein binding of BAA. In the current study, the Lee et al. PBPK model was updated and expanded to include the further metabolism of BAA and the salivary excretion of BE and BAA which may contribute to the forestomach irritation observed in mice in the NTP study. The revised model predicted that peak blood concentrations of BAA achieved following 6 h inhalation exposures are greatest in young adult female rats at concentrations up to 300 ppm. This is not the case predicted for old (> or =18 months) animals, where peak blood concentrations of BAA in male and female mice were similar to or greater than female rats. The revised model serves as a quantitative tool for integrating an extensive pharmacokinetic and mechanistic database into a format that can readily be used to compare internal dosimetry across dose, route of exposure and species.

Development of a physiologically based pharmacokinetic model for ethylene glycol and its metabolite, glycolic Acid, in rats and humans.

An extensive database on the toxicity and modes of action of ethylene glycol (EG) has been developed over the past several decades. Although renal toxicity has long been recognized as a potential outcome, in recent years developmental toxicity, an effect observed only in rats and mice, has become the subject of extensive research and regulatory reviews to establish guidelines for human exposures. The developmental toxicity of EG has been attributed to the intermediate metabolite, glycolic acid (GA), which can become a major metabolite when EG is administered to rats and mice at high doses and dose rates. Therefore, a physiologically based pharmacokinetic (PBPK) model was developed to integrate the extensive mode of action and pharmacokinetic data on EG and GA for use in developmental risk assessments. The resulting PBPK model includes inhalation, oral, dermal, intravenous, and subcutaneous routes of administration. Metabolism of EG and GA were described in the liver with elimination via the kidneys. Metabolic rate constants and partition coefficients for EG and GA were estimated from in vitro studies. Other biochemical constants were optimized from appropriate in vivo pharmacokinetic studies. Several controlled rat and human metabolism studies were used to validate the resulting PBPK model. When internal dose surrogates were compared in rats and humans over a broad range of exposures, it was concluded that humans are unlikely to achieve blood levels of GA that have been associated with developmental toxicity in rats following occupational or environmental exposures.

Validation of human physiologically based pharmacokinetic model for vinyl acetate against human nasal dosimetry data.

Vinyl acetate has been shown to induce nasal lesions in rodents in inhalation bioassays. A physiologically based pharmacokinetic (PBPK) model for vinyl acetate has been used in human risk assessment, but previous in vivo validation was conducted only in rats. Controlled human exposures to vinyl acetate were conducted to provide validation data for the application of the model in humans. Five volunteers were exposed to 1, 5, and 10 ppm 13C1,13C2 vinyl acetate via inhalation. A probe inserted into the nasopharyngeal region sampled both 13C1,13C2 vinyl acetate and the major metabolite 13C1,13C2 acetaldehyde during rest and light exercise. Nasopharyngeal air concentrations were analyzed in real time by ion trap mass spectrometry (MS/MS). Experimental concentrations of both vinyl acetate and acetaldehyde were then compared to predicted concentrations calculated from the previously published human model. Model predictions of vinyl acetate nasal extraction compared favorably with measured values of vinyl acetate, as did predictions of nasopharyngeal acetaldehyde when compared to measured acetaldehyde. The results showed that the current PBPK model structure and parameterization are appropriate for vinyl acetate. These analyses were conducted from 1 to 10 ppm vinyl acetate, a range relevant to workplace exposure standards but which would not be expected to saturate vinyl acetate metabolism. Risk assessment based on this model further concluded that 24 h per day exposures up to 1 ppm do not present concern regarding cancer or non-cancer toxicity. Validation of the vinyl acetate human PBPK model provides support for these conclusions.

Development of an integrated microanalytical system for analysis of lead in saliva and linkage to a physiologically based pharmacokinetic model describing lead saliva secretion.

There is a need to develop reliable portable analytical systems for biomonitoring lead (Pb) in noninvasively collected saliva samples. In addition, appropriate pharmacokinetic analyses are used to quantitate systemic dosimetry based on the saliva Pb concentrations. A portable microfluidics/electrochemical device was developed for the rapid analysis of Pb based on square wave anodic stripping voltammetry, in which a saliva sample flows over an electrode surface, Pb2+ is chemically reduced and accumulated, and the electric potential of the electrode scanned. The system demonstrates a good linear response over a broad Pb concentration range (1-2000 ppb). To evaluate the relationship between saliva and blood Pb, rats were treated with single oral doses ranging from 20 to 500 mg Pb/kg of body weight, and 24 hours later were administered pilocarpine, a muscarinic agonist to induce salivation. To correlate saliva levels with internal dose, blood and saliva were collected and quantitated for Pb by inductively coupled plasma-mass spectrometry (ICP-MS) and by the microanalytical system. The quantitation with the microanalytical system was slightly less (approximately 75-85%) than with ICP-MS; however, the response was linear, with concentration suggesting that it can be used for the quantitation of salivary Pb. To facilitate modeling, a physiologically based pharmacokinetic (PBPK) model for Pb was modified to incorporate a salivary gland compartment. The model was capable of predicting blood and saliva Pb concentration based on a limited data set. These results are encouraging, suggesting that once fully developed the microanalytical system coupled with PBPK modeling can be used as important tools for real-time biomonitoring of Pb for both occupational and environmental exposures.

Uptake, tissue distribution, and fate of inhaled carbon tetrachloride: comparison of rat, mouse, and hamster.

Carbon tetrachloride is hepatotoxic in rats, mice, and hamsters. However, rats are less sensitive to the hepatotoxic effects of CCl(4) than the other two species. The purpose of this study was to compare the uptake, tissue distribution, and elimination of CCl(4) by these three rodent species. Groups of 20 F344/Crl BR rats, B6C3F(1) mice, and Syrian hamsters were exposed by nose-only inhalation for 4 h to 20 ppm (14)C-labeled CCl(4). The fate of (14)C was followed in tissues, excreta, and exhaled breath for 48 h after the exposure. At the end of the exposure, concentrations of CCl(4) equivalents (CE) in tissue were highest in liver of rats and mice, but highest in fat for rats. The liver received the highest dose of CCl(4) equivalents with the following species ranking: mouse > hamster > rat. Patterns of CE elimination were species and tissue dependent, with the majority of elimination occurring within 48 h after exposure. Rats eliminated less radioactivity associated with metabolism ((14)CO(2), urine and feces) and more radioactivity associated with parent compound (exhaled activity trapped on charcoal) than did mice or hamsters. The results indicate that ranking of species sensitivity to the hepatotoxic effects of inhaled CCl(4) correlates with CE dose to liver and with the ability to metabolize CCl(4).

Design and evaluation of a breath-analysis system for biological monitoring of volatile compound.

To ensure the health and safety of workers, integrated industrial hygiene methodologies often include biological monitoring of the workers to help understand their exposure to chemicals. To this end, a field-portable breath-analysis system was developed and tested to measure selected solvents in exhaled air. The exhaled breath data were evaluated using a physiologically based pharmacokinetic (PBPK) model to relate exposure to tissue dose. The system was designed to monitor workers every time they entered or left a work environment--a vast improvement over current 8-hour integrated monitoring strategies. The system combines (1) chemical dosimeters to measure airborne contaminant levels (analyzed in the field/ workplace); (2) real-time breath analysis to quantitate exposure; and 3) PBPK models to estimate internal target tissue dose. To evaluate the system, field tests were conducted at two locations: (1) at an incinerator in Tennessee monitoring benzene and toluene exposures; and (2) a waste repackaging facility in Washington State where hexane, trimethylbenzene, and methylene chloride was monitored. Exhaled breath was sampled and analyzed before and after each specific job task, which ranged from 15 min to 8 hours in duration. In both field studies several volunteers had posttask breath levels higher than pretask levels. The greatest increase corresponded to 573 ppb for methylene chloride and 60 ppb for toluene. Compared with breath analysis, the chemical dosimeters underpredicted the dosimetry, particularly for longer sampling intervals when the volume of air sampled may have diluted exposures. The results of the field studies illustrate the utility of monitoring workers for exposures throughout the day, particularly when job-specific tasks may indicate a potential for exposure.

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

Determination of biokinetic interactions in chemical mixtures using real-time breath analysis and physiologically based pharmacokinetic modeling.

Regulatory agencies are challenged to conduct risk assessments on chemical mixtures without full information on toxicological interactions that may occur at real-world, low-dose exposure levels. The present study was undertaken to investigate the pharmacokinetic impact of low-dose coexposures to toluene and trichloroethylene in vivo in male F344 rats using a real-time breath analysis system coupled with physiologically based pharmacokinetic (PBPK) modeling. Rats were exposed to compounds alone or as a binary mixture, at low (5 to 25 mg/kg) or high (240 to 800 mg/kg) dose levels. Exhaled breath from the exposed animals was monitored for the parent compounds and a PBPK model was used to analyze the data. At low doses, exhaled breath kinetics from the binary mixture exposure compared with those obtained during single exposures, thus indicating that no metabolic interaction occurred with these low doses. In contract, at higher doses the binary PBPK model simulating independent metabolism was found to underpredict the exhaled breath concentration, suggesting an inhibition of metabolism. Therefore the binary mixture PBPK model was used to compare the measured exhaled breath levels from high- and low-dose exposures with the predicted levels under various metabolic interaction simulations (competitive, noncompetitive, or uncompetitive inhibition). Of these simulations, the optimized competitive metabolic interaction description yielded a Ki value closest to the Km of the inhibitor solvent, indicating that competitive inhibition is the most plausible type of metabolic interaction between these two solvents.

Assessment of the percutaneous absorption of trichloroethylene in rats and humans using MS/MS real-time breath analysis and physiologically based pharmacokinetic modeling.

The development and validation of noninvasive techniques for estimating the dermal bioavailability of solvents in contaminated soil and water can facilitate the overall understanding of human health risk. To assess the dermal bioavailability of trichloroethylene (TCE), exhaled breath was monitored in real time using an ion trap mass spectrometer (MS/MS) to track the uptake and elimination of TCE from dermal exposures in rats and humans. A physiologically based pharmacokinetic (PBPK) model was used to estimate total bioavailability. Male F344 rats were exposed to TCE in water or soil under occluded or nonoccluded conditions by applying a patch to a clipper-shaved area of the back. Rats were placed in off-gassing chambers and chamber air TCE concentration was quantified for 3-5 h postdosing using the MS/MS. Human volunteers were exposed either by whole-hand immersion or by attaching patches containing TCE in soil or water on each forearm. Volunteers were provided breathing air via a face mask to eliminate inhalation exposure, and exhaled breath was analyzed using the MS/MS. The total TCE absorbed and the dermal permeability coefficient (K(P)) were estimated for each individual by optimization of the PBPK model to the exhaled breath data and the changing media and/or dermal patch concentrations. Rat skin was significantly more permeable than human skin. Estimates for K(P) in a water matrix were 0.31 +/- 0.01 cm/h and 0.015 +/- 0.003 cm/h in rats and humans, respectively. K(P) estimates were more than three times higher from water than soil matrices in both species. K(P) values calculated using the standard Fick's Law equation were strongly affected by exposure length and volatilization of TCE. In comparison, K(P) values estimated using noninvasive real-time breath analysis coupled with the PBPK model were consistent, regardless of volatilization, exposure concentration, or duration.

A real-time in-vivo method for studying the percutaneous absorption of volatile chemicals.

Realistic estimates of percutaneous absorption following exposures to solvents in the workplace, or through contaminated soil and water, are critical to understanding human health risks. A method was developed to determine dermal uptake of solvents under non-steady-state conditions using real-time breath analysis in rats, monkeys, and humans. The exhaled breath was analyzed using an ion-trap mass spectrometer, which can quantitate chemicals in the exhaled breath stream in the 1-5 ppb range. The resulting data were evaluated using physiologically-based pharmacokinetic (PBPK) models to estimate dermal permeability constants (Kp) under various exposure conditions. The effects of exposure matrix (soil versus water), occlusion versus non-occlusion, and species differences on the absorption of methyl chloroform, trichloroethylene, and benzene were compared. Exposure concentrations were analyzed before and at 0.5-hour intervals throughout the exposures. The percentage of each chemical absorbed and the corresponding Kp were estimated by optimization of the PBPK model to the medium concentration and the exhaled-breath data. The method was found to be sufficiently sensitive for animal and human dermal studies at low exposure concentrations over small body surface areas, for short periods, using non-steady-state exposure conditions.

Utility of real time breath analysis and physiologically based pharmacokinetic modeling to determine the percutaneous absorption of methyl chloroform in rats and humans.

Due to the large surface area of the skin, percutaneous absorption has the potential to contribute significantly to the total bioavailability of some compounds. Breath elimination data, acquired in real-time using a novel MS/MS system, was assessed using a PBPK model with a dermal compartment to determine the percutaneous absorption of methyl chloroform (MC) in rats and humans from exposures to MC in non-occluded soil or occluded water matrices. Rats were exposed to MC using a dermal exposure cell attached to a clipper-shaved area on their back. The soil exposure cell was covered with a charcoal patch to capture volatilized MC and prevent contamination of exhaled breath. This technique allowed the determination of MC dermal absorption kinetics under realistic, non-occluded conditions. Human exposures were conducted by immersing one hand in 0.1% MC in water, or 0.75% MC in soil. The dermal PBPK model was used to estimate skin permeability (Kp) based on the fit of the exhaled breath data. Rat skin K(p)s were estimated to be 0.25 and 0.15 cm/h for MC in water and soil matrices, respectively. In comparison, human permeability coefficients for water matrix exposures were 40-fold lower at 0.006 cm/h. Due to evaporation and differences in apparent Kp, nearly twice as much MC was absorbed from the occluded water (61.3%) compared to the non-occluded soil (32.5%) system in the rat. The PBPK model was used to simulate dermal exposures to MC-contaminated water and soil in children and adults using worst-case EPA default assumptions. The simulations indicate that neither children nor adults will absorb significant amounts of MC from non-occluded exposures, independent of the length of exposure. The results from these simulations reiterate the importance of conducting dermal exposures under realistic conditions.

A compartmental model of water radon contamination in the human body.

A multicompartmental model is developed to simulate the biokinetics in the human body following the ingestion of an inert gas. It was assumed that 100% of the radon ingested appears in the stomach, from which it is transported through the body to different organs via the blood stream. Each of these organs absorbs and releases radon differently, and, amongst all, the fat retains radon for the longest time. To test the model, the information on elimination rates of 222Rn in expired breath was obtained from other experiments done at the University of Maine. The data included male and female participants with a wide range of ages and physical activity levels. The radiation doses in the different organs and tissues were computed. As was expected, the stomach sustained the maximum dose. In this study, the highest stomach dose to any volunteer was evaluated as 115 mSv y(-1).

Evidence of thyroxine formation following iodine administration in Sprague-Dawley rats.

Iodine (I2) has been proposed to be used as a water disinfectant on the manned space station. Previous work has shown that subchronic administration of I2 to Sprague-Dawley rats in drinking water significantly increases plasma thyroxine/triiodothyronine (T4/T3) levels. This is not observed with iodide (I-) treatment. The present study addresses the possibility that I2 reacts with deiodinated T4 metabolites in the gastrointestinal tract to resynthesize T4. Incubation of diiodothyronine (T2), T3, or reverse T3 with I2 in phosphate-buffered saline resulted in the formation of T4 as measured by radioimmunoassay. Washes from the initial segments of the small intestine of the rat show that substrates are present that react with I2 to produce T4. Single oral doses of I2 to rats produced significant dose-related increases in serum T4 and decreases in T3 concentrations after 2 h. Administration of an equivalent dose of I- did not alter significantly plasma T4 concentrations. Higher concentrations of a radioactive substance that bound a T4-specific antibody are present in plasma of animals treated with 125I2 compared to 125I-. These data support the hypothesis that I2 reacts with metabolites of thyroid hormone in the gastrointestinal tract to resynthesize T4 and elevate its levels in blood.

Distribution of iodine into blood components of the Sprague-Dawley rat differs with the chemical form administered.

It has been reported previously that radioactivity derived from iodine distributes differently in the Sprague-Dawley rat depending on the chemical form administered (Thrall and Bull, 1990). In the present communication we report the differential distribution of radioactivity derived from iodine (I2) and iodide (I-) into blood components. Twice as much radioiodine is in the form of I- in the plasma of animals treated with 125I- compared to 125I2-treated rats. No I2 could be detected in the plasma. With an increase in dose, increasing amounts of radioactivity derived from 125I2-treated animals distribute to whole blood compared to equivalent doses of 125I-, reaching a maxima at a dose of 15.8 mumol I/kg body weight. Most of the radioactivity derived from I2 associates with serum proteins and lipids, in particular with albumin and cholesteryl iodide. These data indicate a differential distribution of radioactivity depending on whether it is administered as iodide or iodine. This is inconsistent with the commonly held view that iodine (I2) is reduced to iodide (I-) before it is absorbed systemically from the gastrointestinal tract.

Comparison of toxicity induced by iodine and iodide in male and female rats.

In risk assessments the various forms of iodine have been treated as if they were toxicologically equivalent. While iodide (I-) and iodate (IO3-) have been studied, no studies concerned with the subchronic toxicity of iodine (I2) have been conducted in experimental animals. This study examined toxicities associated with iodine. Rats were treated with 0, 1, 3, 10, and 100 mg/l of either iodine or iodide (as Nal) in the drinking water for 100 d. Treatment had no effect on body, brain, or heart weights in either sex, or on testes weights in male rats. Although differences in kidney and liver weights were noted, they did not appear to be treatment related. Thyroid weight in male rats was significantly increased with an increasing concentration of iodide in the water, but not iodine. In contrast, thyroid weight decreased at the highest dose of iodide in female rats. Hematocrit, hemoglobin, and blood urea nitrogen (BUN) values were relatively constant and did not vary with treatment. There were no significant differences in AST, ALT, cholesterol, and triglyceride values. After 10 d on treatment a dose-related trend in increased plasma T4 concentrations was observed in both sexes treated with iodine. Statistically significant increases in the T4/T3 ratio in both sexes was also noted with iodine treatment. This increase was maintained for 100 d of treatment. Iodide did not produce this effect at 10 d. Although there was a significant increase in T4/T3 ratios in female rats after 100 d of treatment with iodide, the magnitude of the changes was smaller than that observed with iodine treatments. The results of this study indicate that iodine and iodide affect thyroid hormone status in substantially different ways.

Differences in the distribution of iodine and iodide in the Sprague-Dawley rat.

Use of iodine as a drinking water disinfectant for extended space flight raises concerns about potential chronic effects on health. A key question is whether the chemical form of iodine might play a role. To address this question the influence chemical form has on the uptake and distribution of radioiodine was studied in fed and fasted rats. Following oral administration of 125I2 or 125I-, blood 125I levels were maximal at 2 hr and reached similar concentrations in fed animals receiving 125I- and fasted animals receiving either 125I2 or 125I-. However, when 125I2 was administered to fed animals the initial levels of 125I into blood were significantly lower than after the other treatments. The half-life of elimination of 125I from the blood appeared independent of the form of iodine administered. The initial distribution of 125I to the thyroid depended sharply on chemical form, being greater when iodide rather than iodine was administered, whether animals were fed or fasted. In fed animals administered I2, this may largely be explained by the increased retention of 125I in the stomach contents. In fasted animals, both stomach content and blood levels of 125I were similar whether I2 or I- was administered. Since thyroid uptake of iodine is specific for I-, this suggests that the form of iodine in the blood was different in animals administered I2. This notion was further supported by the finding that pretreatment of animals with varying concentrations of I- in drinking water was four times as effective in suppressing the uptake of a test dose of 125I- than pretreatment with equivalent concentrations of I2.