The importance of inclusion of kinetic information in the extrapolation of high-to-low concentrations for human limit setting.

Human health risk assessment of inhalation exposures generally includes a high-to-low concentration extrapolation. Although this is a common step in human risk assessment, it introduces various uncertainties. One of these uncertainties is related to the toxicokinetics. Many kinetic processes such as absorption, metabolism or excretion can be subject to saturation at high concentration levels. In the presence of saturable kinetic processes of the parent compound or metabolites, disproportionate increases in internal blood or tissue concentration relative to the external concentration administered may occur resulting in nonlinear kinetics. The present paper critically reviews human health risk assessment of inhalation exposure. More specific, it emphasizes the importance of kinetic information for the determination of a safe exposure in human risk assessment of inhalation exposures assessed by conversion from a high animal exposure to a low exposure in humans. For two selected chemicals, i.e. methyl tert-butyl ether and 1,2-dichloroethane, PBTK-modelling was used, for illustrative purposes, to follow the extrapolation and conversion steps as performed in existing risk assessments for these chemicals. Human health-based limit values based on an external dose metric without sufficient knowledge on kinetics might be too high to be sufficiently protective. Insight in the actual internal exposure, the toxic agent, the appropriate dose metric, and whether an effect is related to internal concentration or dose is important. Without this, application of assessment factors on an external dose metric and the conversion to continuous exposure results in an uncertain human health risk assessment of inhalation exposures.

Route-to-route extrapolation of 1,2-dichloroethane studies from the oral route to inhalation using physiologically based pharmacokinetic models.

To help develop a comprehensive, quantitative understanding of the hazards of 1,2-dichloroethane (ethylene dichloride, EDC, CAS No. 107-06-2) exposure by the inhalation route, the results of existing subchronic studies and an extended one-generation reproductive toxicity (EOGRT) study recently conducted by the oral route in rats were extrapolated using a physiologically based pharmacokinetic (PBPK) model. The no observed adverse effects levels (NOAELs) for the endpoints of neurotoxicity and reproductive/developmental toxicity were the highest tested doses of 169 and 155 mg/kg-day, respectively. These NOAELs were equivalent to continuous exposure of rats to minimums of 76 ppm and 62 ppm EDC, respectively, using total metabolism of EDC as the dose metric that is equivalent in the oral and inhalation scenarios. In contrast, the subchronic study NOAEL of 37.5 mg/kg-day corresponded to continuous inhalation of 4.4 ppm EDC, based on equivalent extrahepatic metabolism. The selection of the internal metric which serves to establish route-to-route equivalency was found to profoundly influence the NOAEL-equivalent inhalation exposure concentration and thus will be a key determinant of inhalation toxicity reference criteria developed on the basis of EDC studies conducted by the oral route.

Physiologically based pharmacokinetic model development and simulations for ethylene dichloride (1,2-dichloroethane) in rats.

1,2-Dichloroethane (ethylene dichloride, EDC, CAS No. 107-06-2) is a chemical intermediate used in the production of vinyl chloride, trichloroethylene, vinylidene chloride, and trichloroethane. EDC is listed as a Hazardous Air Pollutant (HAP). As such, a need has been identified for a quantitative understanding of the hazards of EDC exposure by the inhalation route. Use of physiologically based pharmacokinetic (PBPK) modeling for route-to-route extrapolation of existing and a future toxicity studies conducted by the oral route may facilitate the quantitative evaluation of potential hazards posed by inhalation of EDC. PBPK models for the disposition of EDC by rats have been previously described, but a need to update the model structure and parameter values was identified based on the current understanding of kinetics of conjugation reactions mediated by glutathione-S-transferases (GSTs) and lack of fit to kinetic data that were not part of the development of previous models. Model structure updates included the addition of extrahepatic metabolism by unspecified enzymes (most likely GSTs or cytochrome P450 enzymes). Chemical-specific disposition parameters were recalibrated and provided good simulations for the majority of the large pharmacokinetic database for single or repeated exposure to EDC via inhalation, gavage, or iv injection in four strains of rats.

Skin penetration and lag times of neat and aqueous diethyl phthalate, 1,2-dichloroethane and naphthalene.

Cutaneous exposures to occupational chemicals may cause toxic effects. For any chemical, the potential for systemic toxicity from dermal exposure depends on its ability to penetrate the skin. Most laboratory studies measure chemical penetration from an aqueous solution through isolated human or laboratory animal skin, although most exposures are not from pure aqueous solutions. The US EPA Interagency Testing Committee (ITC) mandated by the Toxic Substances Control Act, has required industry to measure the in vitro penetration of 34 chemicals in their pure or neat form (if liquid). The goal of the present study was to measure skin permeability and lag time for three neat chemicals of industrial importance, representing the general types of chemicals to be studied by the ITC (non-volatile liquids, volatile liquids, and solids), and to examine interlaboratory variation from these studies. Steady state fluxes and lag times of diethyl phthalate (DEP, slightly volatile), 1,2-dichloroethane (DCE, highly volatile), and naphthalene (NAP, solid) were studied in two different laboratories using different analytical methods. One lab also measured fluxes and lag times from saturated aqueous vehicle. Static diffusion cells, dermatomed hairless guinea pig skin, and gas chromatography were used to measure skin penetration. In the two laboratories, the steady state fluxes (mean+/-SD; microg cm(-2)hour(-1)) of DEP applied neat were: 11.8+/-4.1 and 23.9+/-7.0; fluxes of DCE (neat) were 6280+/-1380 and 3842+/-712; fluxes of NAP from powder were 30.4+/-2.0 and 7.5+/-4.7. Compared with neat fluxes measured in the same laboratory, flux from saturated aqueous solution was higher with DEP (1.9 x) but lower with DCE (0.17 x) and NAP (0.45 x). The three chemicals studied including a dry powder, demonstrate the potential for significant dermal penetration.

Stability and performance of Xanthobacter autotrophicus GJ10 during 1,2-dichloroethane biodegradation.

A nucleic acid-based approach was used to investigate the dynamics of a microbial community dominated by Xanthobacter autotrophicus GJ10 in the degradation of synthetic wastewater containing 1,2-dichloroethane (DCE). This study was performed over a 140-day period in a nonsterile continuous stirred-tank bioreactor (CSTB) subjected to different operational regimens: nutrient-limiting conditions, baseline operation, and the introduction of glucose as a cosubstrate. The microbial community was analyzed by a combination of fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Under nutrient-limiting conditions, DCE degradation was restricted, but this did not affect the dominance of strain GJ10, determined by FISH to comprise 85% of the active population. During baseline operation, DCE degradation improved significantly to over 99.5% and then remained constant throughout the subsequent experimental period. DGGE profiles revealed a stable, complex community, while FISH indicated that strain GJ10 remained the dominant species. During the addition of glucose as a cosubstrate, DGGE profiles showed a proliferation of other species in the CSTB. The percentage of strain GJ10 dropped to 8% of the active population in just 5 days, although this did not affect the DCE biodegradation performance. The return to baseline conditions was accompanied by the reestablishment of strain GJ10 as the dominant species, suggesting that this system responds robustly to external perturbations, both at the functional biodegradation level and at the individual strain level.

Inhibition of cytochrome P-450 with 2-diethylamino-ethyl-2,2-diphenylpropylacetate (SKF-525A) reduces immunotoxicity of chlorinated carbohydrates.

Experiments on outbred albino rats showed that single intraperitoneal injection of cytochrome P-450 inhibitor 2-diethylaminoethyl-2,2-diphenylpropylacetate (SKF-525A) in a dose of 50 mg/kg before acute poisoning with 1,2-dichloroethane and trichloroethane in a dose of 1.0 LD(50), metabolized in the body to compounds with higher toxicity (the phenomenon of "lethal synthesis") reduced their immunotoxicity by decreasing the formation of their biotransformation products.

Determination of pollutant diffusion coefficients in naturally formed biofilms using a single tube extractive membrane bioreactor.

A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of >1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms <1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms >1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (<1-mm thick) biofilms.

Assessment of the developmental toxicity and placental transfer of 1,2-dichloroethane in rats.

This study evaluates the developmental toxicity and placental transfer of 1,2-dichloroethane (DCE) in rats. Sprague-Dawley rats were given 0-2.4 mmol DCE kg-1 day-1 by gavage, or were exposed for 6 hr per day to 0-300 ppm DCE by inhalation, from Day 6 to 20 of gestation. Maternal toxicity was observed after inhalation exposure to 300 ppm DCE and oral administration of 2.0 or 2.4 mmol DCE kg-1. There was no evidence of altered growth nor teratogenic effects after either inhalation or oral administration of DCE at any concentration tested. The time course disposition of 14C was examined over a 48-hr period in 12- and 18-day pregnant rats after a single oral dose of 1.6 mmol [14C]DCE kg-1. Peak concentrations of radiocarbon occurred between 2 and 4 hr postdose. Conceptus (Day 12) and fetal (Day 18) tissues accounted for 0.06 and 0.4% of the administered dose, respectively. Up to 4 hr, levels of radiocarbon in placenta and fetus were slightly less than in maternal plasma of 18-day pregnant rats and were two to five times higher at later periods. At 2 hr, unchanged DCE accounted for most of radioactivity (78-86%) recovered in maternal plasma, placenta, and fetus. Acidic metabolites and radioactivity bound to macromolecules increased up to 24 hr (0.01 mumol-eq DCE g-1) in either placental or fetal tissues. Thereafter, their levels declined more slowly than those in the maternal plasma. Results from this developmental toxicity study in rats confirm embryonic exposure to radiocarbon associated with [14C]DCE and/or its metabolites and has demonstrated the lack of observable teratogenic effects.

Short-term carcinogenesis bioassay of genotoxic procarcinogens in PIM transgenic mice.

E mu-pim-1 transgenic mice, which overexpress the pim-1 oncogene in lymphoid tissues, have shown increased susceptibility to induction of T cell lymphomas by N-ethyl-N-nitrosourea, a direct-acting chemical carcinogen (Nature, 340, 61-63, 1989). We sought to further evaluate E mu-pim-1 transgenic mice as a potential test animal for a short-term carcinogenesis bioassay. We chose to test four genotoxic procarcinogens; 2-acetylaminofluorene (2-AAF), N-nitro-sodiethylamine (NDEA), 1,2-dichloroethane (1,2-DCE) and benzene (BEN). These compounds require metabolic activation and, with the exception of benzene, are not mouse lymphomagens. Compounds were administered by gavage daily for 38 (NDEA and 2-AAF) or 40 (BEN and 1,2-DCE) weeks to groups of 25-29 male and female PIM mice at 1 and 3 mg/kg for NDEA, 50 and 100 mg/kg for BEN, 25-100 mg/kg for 2-AAF and 100-300 mg/kg for 1,2-DCE. Small but statistically significant increases in the incidence of malignant lymphoma were seen for three of the four carcinogens tested; in high dose males treated with 2-AAF, high and low dose females treated with NDEA and high dose females treated with 1,2-DCE. Results for BEN, the only mouse lymphomagen tested, did not show a statistically significant increase in the incidence of malignant lymphomas in transgenic mice within the 40 week duration of the study. NDEA also produced a high incidence (> 70%) of hepatic hemangiosarcomas in both sexes at the low and high dose levels. These results demonstrate that over-expression of the pim-1 oncogene in lymphoid tissue can confer susceptibility of this tissue to chemical carcinogenesis by genotoxic procarcinogens. However, whereas potent genotoxic carcinogens produced only small increases in the incidence of lymphoma and since BEN, a mouse lymphomagen, was negative, PIM transgenic mice may lack sufficient sensitivity to established carcinogens to justify their routine use in a short-term carcinogenesis screening assay.

Gastrointestinal absorption of xenobiotics in physiologically based pharmacokinetic models. A two-compartment description.

Gastrointestinal (GI) absorption in physiologically based pharmacokinetic models is typically described as first-order transfer from one compartment directly into the liver. This approach appears to be adequate for water but not for oily vehicles. In this study, a two-compartment description of GI absorption was developed in which the chemical moved from the first compartment to the second (first-order rate constant KT) and was absorbed from both compartments (first-order rate constants KAS and KAD, respectively) into the liver. Rat blood time-course data sets for methylene chloride, chloroform, dichloroethane, and trichloroethylene after oral gavage in water or corn oil obtained from the literature were used for model validation. Optimization of the KAS, KAD, and KT values for each dosing solution allowed accurate simulation of each data set. In general, the KAS values were 3-4 times greater when water rather than corn oil vehicle was used. The KAD and KT values were similar for the two vehicles. By comparison, a one-compartment description resulted in a poor simulation of the oil gavage data. The two-compartment model rate constant values obtained by optimization of the rat blood time-course data set for trichloroethylene after oral gavage in water were used in the model to predict rat exhaled breath concentrations after oral gavage of trichloroethylene in water (data generated in this laboratory). Exhaled breath trichloroethylene concentrations compared favorably with model predictions.

Physiological model for tissue glutathione depletion and increased resynthesis after ethylene dichloride exposure.

Ethylene dichloride (EDC) is metabolized by two competing pathways both of which consume glutathione (GSH). EDC undergoes oxidation to form chloroacetaldehyde (CAA) which is detoxified by GSH and also reacts directly with GSH to form 2-(s-chloroethyl)-GSH. A physiological pharmacokinetic model developed for EDC was extended to describe tissue GSH turnover and its depletion after EDC exposures. This GSH model was necessary to keep track of GSH concentrations with time, as EDC metabolism is affected by GSH status. Reactions of GSH with EDC and GSH with CAA were defined as second-order. Steady-state GSH formation was modeled as zero-order and GSH loss as first-order. GSH rebound effects after its depletion were controlled by a GSH synthetase reaction, which allowed time- and GSH concentration-dependent feedback for increased GSH resynthesis. The model was developed for liver GSH in the rat and was extrapolated to include the lung. Allometric scaling was used to extrapolate the model to other animal species. Experimental observations in the rat and mouse were consistent with model predictions.