PBTK model for assessment of operator exposure to haloxyfop using human biomonitoring and toxicokinetic data.

Physiologically-based toxicokinetic (PBTK) models are mathematical representations of chemical absorption, distribution, metabolism and excretion (ADME) in animals. Each parameter in a PBTK model describes a physiological, physicochemical or biochemical process that affects ADME. Distributions can be assigned to the model parameters to describe population variability and uncertainty. In this study to assess potential crop sprayer operator exposure to the herbicide haloxyfop, a permeability-limited PBTK model was constructed with parameter uncertainty and variability, and calibrated using Bayesian analysis via Markov chain Monte Carlo methods. A hierarchical statistical model was developed to reconstruct operator exposure using available measurement data: experimentally determined octanol/water partition coefficient, mouse and human toxicokinetic data as well as human biomonitoring data from seven operators who participated in a field study. A chemical risk assessment was performed by comparing the estimated systemic exposure to the acceptable operator exposure level (AOEL). The analysis suggested that in one of the seven operators, the model estimates systemic exposure to haloxyfop of 49.04 ±â€¯10.19 SD µg/kg bw in relation to an AOEL of 5.0 µg/kg bw/day. This does not represent a safety concern as this predicted exposure is well within the 100-fold uncertainty factor applied to the No Observed Adverse Effect Level (NOAEL) in animals. In addition, given the availability of human toxicokinetic data, the 10x uncertainty factor for interspecies differences in ADME could be reduced (EFSA, 2006). Thus the AOEL could potentially be raised tenfold from 5.0 to 50.0 µg/kg bw/day.

Use of a probabilistic PBPK/PD model to calculate Data Derived Extrapolation Factors for chlorpyrifos.

A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model combined with Monte Carlo analysis of inter-individual variation was used to assess the effects of the insecticide, chlorpyrifos and its active metabolite, chlorpyrifos oxon in humans. The PBPK/PD model has previously been validated and used to describe physiological changes in typical individuals as they grow from birth to adulthood. This model was updated to include physiological and metabolic changes that occur with pregnancy. The model was then used to assess the impact of inter-individual variability in physiology and biochemistry on predictions of internal dose metrics and quantitatively assess the impact of major sources of parameter uncertainty and biological diversity on the pharmacodynamics of red blood cell acetylcholinesterase inhibition. These metrics were determined in potentially sensitive populations of infants, adult women, pregnant women, and a combined population of adult men and women. The parameters primarily responsible for inter-individual variation in RBC acetylcholinesterase inhibition were related to metabolic clearance of CPF and CPF-oxon. Data Derived Extrapolation Factors that address intra-species physiology and biochemistry to replace uncertainty factors with quantitative differences in metrics were developed in these same populations. The DDEFs were less than 4 for all populations. These data and modeling approach will be useful in ongoing and future human health risk assessments for CPF and could be used for other chemicals with potential human exposure.

Dose-Response for Multiple Biomarkers of Exposure and Genotoxic Effect Following Repeated Treatment of Rats with the Alkylating Agents, MMS and MNU.

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

Development of a physiologically-based pharmacokinetic model of 2-phenoxyethanol and its metabolite phenoxyacetic acid in rats and humans to address toxicokinetic uncertainty in risk assessment.

2-Phenoxyethanol (PhE) has been shown to induce hepatotoxicity, renal toxicity, and hemolysis at dosages ≥ 400 mg/kg/day in subchronic and chronic studies in multiple species. To reduce uncertainty associated with interspecies extrapolations and to evaluate the margin of exposure (MOE) for use of PhE in cosmetics and baby products, a physiologically-based pharmacokinetic (PBPK) model of PhE and its metabolite 2-phenoxyacetic acid (PhAA) was developed. The PBPK model incorporated key kinetic processes describing the absorption, distribution, metabolism and excretion of PhE and PhAA following oral and dermal exposures. Simulations of repeat dose rat studies facilitated the selection of systemic AUC as the appropriate dose metric for evaluating internal exposures to PhE and PhAA in rats and humans. Use of the PBPK model resulted in refinement of the total default UF for extrapolation of the animal data to humans from 100 to 25. Based on very conservative assumptions for product composition and aggregate product use, model-predicted exposures to PhE and PhAA resulting from adult and infant exposures to cosmetic products are significantly below the internal dose of PhE observed at the NOAEL dose in rats. Calculated MOEs for all exposure scenarios were above the PBPK-refined UF of 25.

A novel approach for concurrent quantitation of glutathione, glutathione disulfide, and 2-hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide, using liquid chromatography-positive electrospray tandem mass spectrometry.

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.

Chlorpyrifos PBPK/PD model for multiple routes of exposure.

1. Chlorpyrifos (CPF) is an important pesticide used to control crop insects. Human Exposures to CPF will occur primarily through oral exposure to residues on foods. A physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model has been developed that describes the relationship between oral, dermal and inhalation doses of CPF and key events in the pathway for cholinergic effects. The model was built on a prior oral model that addressed age-related changes in metabolism and physiology. This multi-route model was developed in rats and humans to validate all scenarios in a parallelogram design. 2. Critical biological effects from CPF exposure require metabolic activation to CPF oxon, and small amounts of metabolism in tissues will potentially have a great effect on pharmacokinetics and pharmacodynamic outcomes. Metabolism (bioactivation and detoxification) was therefore added in diaphragm, brain, lung and skin compartments. Pharmacokinetic data are available for controlled human exposures via the oral and dermal routes and from oral and inhalation studies in rats. The validated model was then used to determine relative dermal versus inhalation uptake from human volunteers exposed to CPF in an indoor scenario.

A human life-stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: development and validation.

Sensitivity to some chemicals in animals and humans are known to vary with age. Age-related changes in sensitivity to chlorpyrifos have been reported in animal models. A life-stage physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model was developed to predict disposition of chlorpyrifos and its metabolites, chlorpyrifos-oxon (the ultimate toxicant) and 3,5,6-trichloro-2-pyridinol (TCPy), as well as B-esterase inhibition by chlorpyrifos-oxon in humans. In this model, previously measured age-dependent metabolism of chlorpyrifos and chlorpyrifos-oxon were integrated into age-related descriptions of human anatomy and physiology. The life-stage PBPK/PD model was calibrated and tested against controlled adult human exposure studies. Simulations suggest age-dependent pharmacokinetics and response may exist. At oral doses ⩾0.6mg/kg of chlorpyrifos (100- to 1000-fold higher than environmental exposure levels), 6months old children are predicted to have higher levels of chlorpyrifos-oxon in blood and higher levels of red blood cell cholinesterase inhibition compared to adults from equivalent doses. At lower doses more relevant to environmental exposures, simulations predict that adults will have slightly higher levels of chlorpyrifos-oxon in blood and greater cholinesterase inhibition. This model provides a computational framework for age-comparative simulations that can be utilized to predict chlorpyrifos disposition and biological response over various postnatal life stages.

Toxicokinetic profile of N-(2-aminoethyl)ethanolamine in the female Wistar rat and distribution into the late gestation fetus and milk.

N-(2-aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t(1/2) = 0.1-0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t(1/2 α) = 1.6-1.8 hr) followed by a slower phase (t(1/2 ß) = 16.7-17.3 hr). Following repeated gavage administration during gestation day 17 to 19, (14) C-AEEA-derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, (14) C-AEEA-derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6- and 2.5-fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.

TK Modeler version 1.0, a Microsoft® Excel®-based modeling software for the prediction of diurnal blood/plasma concentration for toxicokinetic use.

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies.

Statistical methodology to determine kinetically derived maximum tolerated dose in repeat dose toxicity studies.

Several statistical approaches were evaluated to identify an optimum method for determining a point of nonlinearity (PONL) in toxicokinetic data. (1) A second-order least squares regression model was fit iteratively starting with data from all doses. If the second order term was significant (α<0.05), the dataset was reevaluated with successive removal of the highest dose until the second-order term became non-significant. This dose, whose removal made the second order term non-significant, is an estimate of the PONL. (2) A least squares linear model was fit iteratively starting with data from all doses except the highest. The mean response for the omitted dose was compared to the 95% prediction interval. If the omitted dose falls outside the confidence interval it is an estimate of the PONL. (3) Slopes of least squares linear regression lines for sections of contiguous doses were compared. Nonlinearity was suggested when slopes of compared sections differed. A total of 33 dose-response datasets were evaluated. For these toxicokinetic data, the best statistical approach was the least squares regression analysis with a second-order term. Changing the α level for the second-order term and weighting the second-order analysis by the inverse of feed consumption were also considered. This technique has been shown to give reproducible identification of nonlinearities in TK datasets.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

Use of toxicokinetics to support chemical evaluation: Informing high dose selection and study interpretation.

Toxicokinetic (TK) information can substantially enhance the value of the data generated from toxicity testing, and is an integral part of pharmaceutical safety assessment. It is less widely used in the chemical, agrochemical and consumer products industries, but recognition of its value is growing, as reflected by increased reference to the use of TK information in new and draft OECD test guidelines. To help promote increased consideration of the important role TK can play in chemical risk assessment, we have gathered practical examples from the peer-reviewed literature, as well as in-house industry data, that highlight opportunities for the use of TK in the selection of dose levels. Use of TK can help to ensure studies are designed to be of most relevance to assessing potential risk in humans, and avoid the use of excessively high doses that could result in unnecessary suffering in experimental animals. Greater emphasis on the potential contribution of TK in guiding study design and interpretation should be incorporated in regulatory data requirements and associated guidance.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

In vitro age-dependent enzymatic metabolism of chlorpyrifos and chlorpyrifos-oxon in human hepatic microsomes and chlorpyrifos-oxon in plasma.

Age-dependent chlorpyrifos (CPF) metabolism was quantified by in vitro product formation in human hepatic microsomes (ages 13 days to 75 years) and plasma (ages 3 days to 43 years) with gas chromatography-mass spectrometry. Hepatic CPF cytochrome P450 desulfuration [CPF to chlorpyrifos-oxon (CPF-oxon)] and dearylation (CPF to 3,5,6-trichloro-2-pyridinol) V(max) values were 0.35 ± 0.21 and 0.73 ± 0.38 nmol · min(-1) · mg microsomal protein (-1) (mean ± S.D.), respectively. The mean (±S.D.) hepatic CPF-oxon hydrolysis (chlorpyrifos-oxonase [CPFOase]) V(max) was 78 ± 44 nmol · min(-1) · mg microsomal protein (-1). None of these hepatic measures demonstrated age-dependent relationships on a per microsomal protein basis using linear regression models. Ratios of CPF bioactivation to detoxification (CPF desulfuration to dearylation) V(max) values were consistent across ages. CPFOase in plasma demonstrated age-dependent increases on a volume of plasma basis, as did total plasma protein levels. Mean (±S.D.) CPF-oxon hydrolysis V(max) values for children <6 months of age and adults (≥16 years) were 1900 ± 660 and 6800 ± 1600 nmol · min(-1) · ml(-1), respectively, and at environmental exposure levels, this high- capacity enzyme is likely to be sufficient even in infants. Plasma samples were phenotyped for paraoxonase status, and frequencies were 0.5, 0.4, and 0.1 for QQ, QR, and RR phenotypes, respectively. These results will be integrated into a physiologically based pharmacokinetic and pharmacodynamic model for CPF and, once integrated, will be useful for assessing biological response to CPF exposures across life stages.

In vitro metabolism, glutathione conjugation, and CYP isoform specificity of epoxidation of 4-vinylphenol.

4-Vinylphenol (4VP) has been identified as a minor urinary metabolite of styrene in rat and human volunteers. This compound has been shown to be more hepatotoxic and pneumotoxic than both styrene and styrene oxide at lower doses in rats and mice. To explore the possible toxicity mechanism of 4VP, the current study was conducted to investigate the metabolism of 4VP, the glutathione (GSH) conjugation of the metabolites of 4VP and its cytochrome P(450) (CYP) specificity in epoxidation in different microsomes in vitro. Incubations of 4VP with mouse lung microsomes afforded two major metabolites which were identified as 4-(2-oxiranyl)-phenol of 4VP (4VPO) and 4VP catechol. 4VPO was found to react with GSH to form GSH conjugate and 4VP catechol was found to further be metabolized to electrophilic species which react with GSH to form the corresponding 4VP catechol GSH conjugates. Relative formation rates for those GSH conjugates and the regioisomer formation of 4VPO-GSH conjugates with both inhibitors of CYP 2F2 and CYP 2E1 in microsomal incubation condition were also investigated. This present study provides better insight on the lung toxicity seen with 4VP, the toxic metabolite of commercial styrene.

Development of a source-to-outcome model for dietary exposures to insecticide residues: an example using chlorpyrifos.

Probabilistic models of interindividual variation in exposure and response were linked to create a source-to-outcome population model. This model was used to investigate cholinesterase inhibition from dietary exposures to an insecticide (chlorpyrifos) in populations of adults and 3 year old children. A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model was used to calculate the variation in sensitivity occurring from interindividual variability in physiology, metabolism, and physical activity levels. A dietary intake model characterizes the variation in dietary insecticide exposures and variation in anthropometry in the populations. Published equations were used to describe the necessary physiology for each simulated individual based on the anthropometry from the dietary intake model. The model of the interindividual variation in response to chlorpyrifos was developed by performing a sensitivity analysis on the PBPK/PD model to determine the parameters that drive variation in pharmacodynamics outcomes (brain and red blood cell acetylcholinesterase inhibition). Distributions of interindividual variation were developed for parameters with the largest impact; the probabilistic model sampled from these distributions. The impact of age and interindividual variation on sensitivity at the doses that occur from dietary exposures, typically orders of magnitude lower than exposures assessed in toxicological studies, was assessed using the source-to-outcome model. The resulting simulations demonstrated that metabolic detoxification capacity was sufficient to prevent significant brain and red blood cell acetylcholinesterase inhibition, even in individuals with the lowest detoxification potential. Age-specific pharmacokinetic and pharmacodynamic parameters did not predict differences in susceptibility between adults and children. In the future, the approach of this case study could be used to assess the risks from low level exposures to other chemicals.

Application of a source-to-outcome model for the assessment of health impacts from dietary exposures to insecticide residues.

The paper presents a case study of the application of a "source-to-outcome" model for the evaluation of the health outcomes from dietary exposures to an insecticide, chlorpyrifos, in populations of adults (age 30) and children (age 3). The model is based on publically-available software programs that characterize the longitudinal dietary exposure and anthropometry of exposed individuals. These predictions are applied to a validated PBPK/PD model to estimate interindividual and longitudinal variation in brain and RBC AChE inhibition (key events) and chlorpyrifos concentrations in blood and TCPy in urine (biomarkers of exposure). The predicted levels of chlorpyrifos and TCPy are compared to published measurements of the biomarkers. Predictions of RBC AChE are compared to levels of inhibition associated with reported exposure-related effects in humans to determine the potential for the occurrence of adverse cholinergic effects. The predicted distributions of chlorpyrifos in blood and TCPy in urine were found to be reasonably consistent with published values, supporting the predictive value of the exposure and PBPK portions of the source-to-outcome model. Key sources of uncertainty in predictions of dietary exposures were investigated and found to have a modest impact on the model predictions. Future versions of this source-to-outcome model can be developed that consider advances in our understanding of metabolism, to extend the approach to other age groups (infants), and address intakes from other routes of exposure.

In vitro metabolism and covalent binding of ethylbenzene to microsomal protein as a possible mechanism of ethylbenzene-induced mouse lung tumorigenesis.

This study was conducted to determine species differences in covalent binding of the reactive metabolites of ethylbenzene (EB) formed in the liver and lung microsomes of mouse, rat and human in the presence of NADPH. These data further the understanding of the mechanism by which EB causes mouse specific lung toxicity and a follow-up to our earlier report of the selective elevation, although minor, of the ring-oxidized reactive metabolites in mouse lung microsomes (Saghir et al., 2009). Binding assays were also conducted with or without 5-phenyl-1-pentyne (5P1P), an inhibitor of CYP 2F2, and diethyldithiocarbamate (DDTC), an inhibitor of CYP 2E1 to evaluate their role in the formation of the related reactive metabolites. Liver and lung microsomes were incubated with (14)C-EB (0.22 mM) in the presence of 1mM NADPH under physiological conditions for 60 min. In lung microsomes, binding activity was in the order of mouse (812.4+/-102.2 pmol/mg protein)>>rat (57.0+/-3.2 pmol/mg protein). Human lung microsomes had little binding activity (15.7+/-1.4 pmol/mg protein), which was comparable to the no-NADPH control (9.9-16.7 pmol/mg protein). In liver microsomes, mouse had the highest activity (469.0+/-38.5 pmol/mg protein) followed by rat (148.3+/-14.7 pmol/mg protein) and human (89.8+/-3.0 pmol/mg protein). Presence of 5P1P or DDTC decreased binding across species and tissues. However, much higher inhibition was observed in mouse (86% [DDTC] and 89% [5P1P]) than rat (56% [DDTC] and 59% [5P1P]) lung microsomes. DDTC showed approximately 2-fold higher inhibition of binding in mouse and human liver microsomes than 5P1P (mouse=85% vs. 40%; human=59% vs. 36%). Inhibition in binding by DDTC was much higher (10-fold) than 5P1P (72% vs. 7%) in rat liver microsomes. These results show species, tissue and enzyme differences in the formation of reactive metabolites of EB. In rat and mouse lung microsomes, both CYP2E1 and CYP2F2 appear to contribute in the formation of reactive metabolites of EB. In contrast, CYP2E1 appears to be the primary CYP isozyme responsible for the reactive metabolites of EB in the liver.

Dermal penetration of ethylene glycol through human skin in vitro.

This study was conducted to determine the in vitro dermal absorption of ethylene glycol (EG) through dermatomed human abdominal skin (containing epidermis and dermis), obtained from cadavers within 24 hours of death and kept frozen until processed. Three formulations of EG (neat, 50%, and 10% aqueous solutions) were applied in triplicate to skin samples from 6 donors, and placed in Teflon Bronaugh flow-through diffusion cells. Barrier integrity of each sample was evaluated with (3)H-H(2)O prior to applying EG and only data from samples passing the test were used. A physiological receptor fluid was pumped beneath the skin samples and collected in a fraction collector at predetermined time points through 24 hours. Possible volatilized EG was trapped in a charcoal basket located above each skin sample. Each skin sample was treated with an infinite dose of 500 microL of EG formulation/cm(2). At the end of 24 hours, volatilized EG trapped in the headspace was collected, the unabsorbed dose was removed from the skin and the skin was rinsed, tape stripped, and solubilized along with a rinse of the flow-through cells, and total radioactivity was determined. Only a small fraction (