P. Robinan Gentry

Evaluation of the Uncertainty in an Oral Reference Dose for Methylmercury Due to Interindividual Variability in Pharmacokinetics

An analysis of the uncertainty in guidelines for the ingestion of methylmercury (MeHg) due to human pharmacokinetic variability was conducted using a physiologically based pharmacokinetic (PBPK) model that describes MeHg kinetics in the pregnant human and fetus. Two alternative derivations of an ingestion guideline for MeHg were considered: the U.S. Environmental Protection Agency reference dose (RfD) of 0.1 microgram/kg/day derived from studies of an Iraqi grain poisoning episode, and the Agency for Toxic Substances and Disease Registry chronic oral minimal risk level (MRL) of 0.5 microgram/kg/day based on studies of a fish-eating population in the Seychelles Islands. Calculation of an ingestion guideline for MeHg from either of these epidemiological studies requires calculation of a dose conversion factor (DCF) relating a hair mercury concentration to a chronic MeHg ingestion rate. To evaluate the uncertainty in this DCF across the population of U.S. women of child-bearing age, Monte Carlo analyses were performed in which distributions for each of the parameters in the PBPK model were randomly sampled 1000 times. The 1st and 5th percentiles of the resulting distribution of DCFs were a factor of 1.8 and 1.5 below the median, respectively. This estimate of variability is consistent with, but somewhat less than, previous analyses performed with empirical, one-compartment pharmacokinetic models. The use of a consistent factor in both guidelines of 1.5 for pharmacokinetic variability in the DCF, and keeping all other aspects of the derivations unchanged, would result in an RfD of 0.2 microgram/kg/day and an MRL of 0.3 microgram/kg/day.

Evaluation of the potential impact of pharmacokinetic differences on tissue dosimetry in offspring during pregnancy and lactation

In recent years efforts have increased to develop a framework for assessing differences, both pharmacokinetic and pharmacodynamic, between children and adults for purposes of assessing risk of adverse effects following chemical exposure. The specific goal of this study was to demonstrate an approach for using PBPK modeling to compare maternal and fetal/neonatal blood and tissue dose metrics during pregnancy and lactation. Six chemical classes were targeted to provide a variety of physicochemical properties (volatility, lipophilicity, water solubility), and surrogate chemicals were selected to represent each class (isopropanol, vinyl chloride, methylene chloride, perchloroethylene, nicotine, and TCDD), based on the availability of pharmacokinetic information. These chemicals were also selected to provide different pharmacokinetic characteristics, including metabolic production of stable or reactive intermediates in the liver and competing pathways for metabolism. Changes in dosimetry during pregnancy predicted by the modeling were mainly attributable to the development of enzymatic pathways in the fetus or to changes in tissue composition in the mother and fetus during pregnancy. In general, blood concentrations were lower in the neonate during the lactation period than in the fetus during gestation. This postnatal decrease varied from only a slight change (for TCDD) to approximately four orders of magnitude (for vinyl chloride). As compared to maternal exposure, fetal/neonatal exposures ranged from approximately twice as great (for TCDD) to several orders of magnitude lower (for isopropanol). The results of this study are in general agreement with the analyses of data on pharmaceutical chemicals, which have suggested that the largest difference in pharmacokinetics observed between children and adults is for the perinatal period. The most important factor appears to be the potential for decreased clearance of toxic chemicals in the perinatal period due to immature metabolic enzyme systems, although this same factor can also reduce the risk from reactive metabolites during the same period.

Evaluation of the Potential Impact of Age- and Gender-Specific Lung Morphology and Ventilation Rate on the Dosimetry of Vapors

In recent years, there have been growing concerns that due to differences, both pharmacokinetic and pharmacodynamic, between children and adults, children could be at greater risk of adverse effects following chemical exposure. The specific goal of this study was to demonstrate an approach for using physiologically based pharmacokinetic (PBPK) modeling to compare inhalation dosimetry in the adult and the child of both males and females. Three categories of gases were considered: rapidly and irreversibly reactive in the respiratory tract (ozone), relatively water-soluble and nonreactive (isopropanol), and relatively water-insoluble and nonreactive (styrene, vinyl chloride, and perchloroethylene). The nonreactive chemicals were also selected because they are metabolized in the respiratory tract. The age-related changes observed for the estimated dose metrics were a function of the physiochemical properties of the inhaled vapor and their interactions in the body. Blood concentrations estimated for all vapors, either poorly metabolized (e.g., PERC), moderately metabolized (e.g., ST), or highly metabolized vapors (e.g., IPA and VC), varied less than a factor of two between infants and adults. These changes, moreover, were confined to the first year after birth, a relatively short window compared to the total lifespan of the individual. In contrast, circulating metabolite concentrations estimated in the blood, as well as amounts metabolized in the liver and lung, appeared to be a strong function of age, due to their dependence on the maturity of the pertinent metabolic enzyme systems.

Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicity.

A comprehensive literature search was conducted to identify information on gene expression changes following exposures to inorganic arsenic compounds. This information was organized by compound, exposure, dose/concentration, species, tissue, and cell type. A concentration‐related hierarchy of responses was observed, beginning with changes in gene/protein expression associated with adaptive responses (e.g., preinflammatory responses, delay of apoptosis). Between 0.1 and 10 μM, additional gene/protein expression changes related to oxidative stress, proteotoxicity, inflammation, and proliferative signaling occur along with those related to DNA repair, cell cycle G2/M checkpoint control, and induction of apoptosis. At higher concentrations (10–100 μM), changes in apoptotic genes dominate. Comparisons of primary cell results with those obtained from immortalized or tumor‐derived cell lines were also evaluated to determine the extent to which similar responses are observed across cell lines. Although immortalized cells appear to respond similarly to primary cells, caution must be exercised in using gene expression data from tumor‐derived cell lines, where inactivation or overexpression of key genes (e.g., p53, Bcl‐2) may lead to altered genomic responses. Data from acute in vivo exposures are of limited value for evaluating the dose‐response for gene expression, because of the transient, variable, and uncertain nature of tissue exposure in these studies. The available in vitro gene expression data, together with information on the metabolism and protein binding of arsenic compounds, provide evidence of a mode of action for inorganic arsenic carcinogenicity involving interactions with critical proteins, such as those involved in DNA repair, overlaid against a background of chemical stress, including proteotoxicity and depletion of nonprotein sulfhydryls. The inhibition of DNA repair under conditions of toxicity and proliferative pressure may compromise the ability of cells to maintain the integrity of their DNA. Environ. Mol. Mutagen., 2010.

Evaluation of Physiologically Based Pharmacokinetic Models in Risk Assessment: An Example with Perchloroethylene

One of the more problematic aspects of the application of physiologically based pharmacokinetic (PBPK) models in risk assessment is the question of whether the model has been adequately validated to provide confidence in the dose metrics calculated with it. A number of PBPK models have been developed for perchloroethylene (PCE), differing primarily in the parameters estimated for metabolism. All of the models provide reasonably accurate simulations of selected kinetic data for PCE in mice and humans and could thus be considered to be “validated” to some extent. However, quantitative estimates of PCE cancer risk are critically dependent on the prediction of the rate of metabolism at low environmental exposures. Recent data on the urinary excretion of trichloroacetic acid (TCA), the major metabolite of PCE, for human subjects exposed to lower concentrations than those used in previous studies, make it possible to compare the high- to low-dose extrapolation capability of the various published human models. The model of Gearhart et al., which is the only model to include a description of TCA kinetics, provided the closest predictions of the urinary excretion observed in these low-concentration exposures. Other models overestimated metabolite excretion in this study by 5- to 15-fold. A systematic discrepancy between model predictions and experimental data for the time course of the urinary excretion of TCA suggested a contribution from TCA formed by metabolism of PCE in the kidney and excreted directly into the urine. A modification of the model of Gearhart et al. to include metabolism of PCE to TCA in the kidney at 10% of the capacity of the liver, with direct excretion of the TCA formed in the kidney into the urine, markedly improved agreement with the experimental time-course data, without altering predictions of liver metabolism. This case study with PCE demonstrates the danger of relying on parent chemical kinetic data to validate a model that will be used for the prediction of metabolism.

Physiologically Based Pharmacokinetic Modeling of Arsenic in the Mouse

A remarkable feature of the carcinogenicity of inorganic arsenic is that while human exposures to high concentrations of inorganic arsenic in drinking water are associated with increases in skin, lung, and bladder cancer, inorganic arsenic has not typically caused tumors in standard laboratory animal test protocols. Inorganic arsenic administered for periods of up to 2 yr to various strains of laboratory mice, including the Swiss CD-1, Swiss CR:NIH(S), C57Bl/6p53(+/−), and C57Bl/6p53(+/+), has not resulted in significant increases in tumor incidence. However, Ng et al. (1999) have reported a 40% tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. In order to investigate the potential role of tissue dosimetry in differential susceptibility to arsenic carcino-genicity, a physiologically based pharmacokinetic (PBPK) model for inorganic arsenic in the rat, hamster, monkey, and human (Mann et al., 1996a, 1996) was extended to describe the kinetics in the mouse. The PBPK model was parameterized in the mouse using published data from acute exposures of B6C3F1 mice to arsenate, arsenite, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) and validated using data from acute exposures of C57Black mice. Predictions of the acute model were then compared with data from chronic exposures. There was no evidence of changes in the apparent volume of distribution or in the tissue-plasma concentration ratios between acute and chronic exposure that might support the possibility of inducible arsenite efflux. The PBPK model was also used to project tissue dosimetry in the C57Bl/6J study, in comparison with tissue levels in studies having shorter duration but higher arsenic treatment concentrations. The model evaluation indicates that pharmacokinetic factors do not provide an explanation for the difference in outcomes across the various mouse bioassays. Other possible explanations may relate to strain-specific differences, or to the different durations of dosing in each of the mouse studies, given the evidence that inorganic arsenic is likely to be active in the later stages of the carcinogenic process.

Determination of a site-specific reference dose for methylmercury for fish-eating populations

Environmental risk-management decisions in the U.S. involving potential exposures to methylmercury currently use a reference dose (RfD) developed by the U.S. Environmental Protection Agency (USEPA). This RfD is based on retrospective studies of an acute poisoning incident in Iraq in which grain contaminated with a methylmercury fungicide was inadvertently used in the baking of bread.1 The exposures, which were relatively high but lasted only a few months, were associated with neurological effects in both adults (primarily paresthesia) and infants (late walking, late talking, etc.). It is generally believed that the developing fetus represents a particularly sensitive subpopulation for the neurological effects of methylmercury. The USEPA derived an RfD of 0.1 g/kg/day based on benchmark dose (BMD) modeling of the combined neurological endpoints reported for children exposed in utero. This RfD included an uncertainty factor of 10 to consider human pharmacokinetic variability and database limitations (lack of data on multigeneration effects or possible long-term sequelae of perinatal exposure). Alcoa signed an Administrative Order of Consent for the conduct of a remedial investigation/feasibility study (RI/FS) at their Point Comfort Operations and the adjacent Lavaca Bay in Texas to address the effects of historical discharges of mercury-containing wastewater. In cooperation with the Texas Natural Resource Conservation Commission and USEPA Region VI, Alcoa conducted a baseline risk assessment to assess potential risk to human health and the environment. As a part of this assessment, Alcoa pursued the development of a site-specific RfD for methylmercury to specifically address the potential human health effects associated with the ingestion of contaminated finfish and shellfish from Lavaca Bay. Application of the published USEPA RfD to this site is problematic; while the study underlying the RfD represented acute exposure to relatively high concentrations of methylmercury, the exposures of concern for the Point Comfort site are from the chronic consumption of relatively low concentrations of methylmercury in fish. Since the publication of the USEPA RfD, several analyses of chronic exposure to methylmercury in fish-eating populations have been reported. The purpose of the analysis reported here was to evaluate the possibility of deriving an RfD for methylmercury, specifically for the case of fish ingestion, on the basis of these new studies. In order to better support the risk-management decisions associated with developing a remediation approach for the site in question, the analysis was designed to provide information on the distribution of acceptable ingestion rates across a population, which could reasonably be expected to be consistent with the results of the epidemiological studies of other fish-eating populations. Based on a review of the available literature on the effects of methylmercury, a study conducted with a population in the Seychelles Islands was selected as the critical study for this analysis. The exposures to methylmercury in this population result from chronic, multigenerational ingestion of contaminated fish. This prospective study was carefully conducted and analyzed, included a large cohort of mother-infant pairs, and was relatively free of confounding factors. The results of this study are essentially negative, and a no-observed-adverse-effect level (NOAEL) derived from the estimated exposures has recently been used by the Agency for Toxic Substances and Disease Registry (ATSDR) as the basis for a chronic oral minimal risk level (MRL) for methylmercury. In spite of the fact that no statistically significant effects were observed in this study, the data as reported are suitable for dose-response analysis using the BMD method. Evaluation of the BMD method used in this analysis, as well as in the current USEPA RfD, has demonstrated that the resulting 95% lower bound on the 10% benchmark dose (BMDL) represents a conservative estimate of the traditional NOAEL, and that it is superior to the use of “average” or “grouped” exposure estimates when dose-response information is available, as is the case for the Seychelles study. A more recent study in the Faroe Islands, which did report statistically significant associations between methylmercury exposure and neurological effects, could not be used for dose-response modeling due to inadequate reporting of the data and confounding from co-exposure to polychlorinated biphenyls (PCBs). BMD modeling over the wide range of neurological endpoints reported in the Seychelles study yielded a lowest BMDL for methylmercury in maternal hair of 21 ppm. This BMDL was then converted to an expected distribution of daily ingestion rates across a population using Monte Carlo analysis with a physiologically based pharmacokinetic (PBPK) model to evaluate the impact of interindividual variability. The resulting distribution of ingestion rates at the BMDL had a geometric mean of 1.60 g/kg/day with a geometric standard deviation of 1.33; the 1st, 5th, and 10th percentiles of the distribution were 0.86, 1.04, and 1.15 g/kg/day. In place of the use of an uncertainty factor of 3 for pharmacokinetic variability, as is done in the current RfD, one of these lower percentiles of the daily ingestion rate distribution provides a scientifically based, conservative basis for taking into consideration the impact of pharmacokinetic variability across the population. On the other hand, it was felt that an uncertainty factor of 3 for database limitations should be used in the current analysis. Although there can be high confidence in the benchmark-estimated NOAEL of 21 ppm in the Seychelles study, some results in the New Zealand and Faroe Islands studies could be construed to suggest the possibility of effects at maternal hair concentrations below 10 ppm. In addition, while concerns regarding the possibility of chronic sequelae are not supported by the available data, neither can they be absolutely ruled out. The use of an uncertainty factor of 3 is equivalent to using a NOAEL of 7 ppm in maternal hair, which provides additional protection against the possibility that effects could occur at lower concentrations in some populations. Based on the analysis described above, the distribution of acceptable daily ingestion rates (RfDs) recommended to serve as the basis for site-specific risk-management decisions at Alcoa’s Point Comfort Operations ranges from approximately 0.3 to 1.1 g/kg/day, with a population median (50th percentile) of 0.5 g/kg/day. By analogy with USEPA guidelines for the use of percentiles in applications of distributions in exposure assessments, the 10th percentile provides a reasonably conservative measure. On this basis, a site-specific RfD of 0.4 g/kg/day is recommended.

Data for Physiologically Based Pharmacokinetic Modeling in Neonatal Animals: Physiological Parameters in Mice and Sprague-Dawley Rats

ABSTRACTRecent scientific and policy initiatives have resulted in increased interest in risk to fetuses, infants, and children and consideration of how such risks should be evaluated. A useful way of addressing this issue is to use physiologically based pharmacokinetic (PBPK) models to compare the tissue dose that children and adults receive for a given amount of a chemical ingested or inhaled. The response in children and adults for a given tissue dose can also be compared. To aid in the development of age-specific PBPK models for experimental animals, we have collected information on physiological parameters in neonates and young animals, through 60 days of age. Our effort focused on generic physiological values, such as tissue weight (termed tissue volume in the context of PBPK modeling), intake (alveolar ventilation, food intake, water intake), and flows (blood flows to tissues, bile flow, creatinine clearance, and glomerular filtration rate). To date, parameters for Sprague-Dawley rats and mice of mu...

Requirements for a biologically realistic cancer risk assessment for inorganic arsenic

A remarkable feature of the carcinogenicity of inorganic arsenic (As,) is the observation that human exposures to Asi have been strongly associated with increases in skin, lung, and internal cancers, but As, does not typically cause tumors in standard laboratory animal test protocols. Considerable controversy has centered on whether there is epidemiological evidence of a “threshold” for the carcinogenic effects of Asi, or at least of a highly nonlinear dose–response. Saturation of metabolism in the dose-range associated with tumors does not appear to be adequate to produce a major impact on the dose-response for carcinogenicity. If there is a strong nonlinearity, it results from the nature of the carcinogenic mechanism(s) of Asi. However, no single hypothesis for the mechanism of Asi carcinogenicity has widespread support. A biologically realistic cancer risk assessment for Asi would requirea quantitative description of the dose of active arsenic species in target tissues, the interactions between active arsenic and tissue constituents, and the manner in which these interactions result in tumor formation in multiple organs in humans, but not in experimental animals. Although Asi has only infrequently been associated with tumors in animal studies, it has repeatedly been shown to act as a comutagen in vitro and as a cocarcinogen in vivo. Asi is clastogenic, producing chromatid aberrations, but does not produce point mutations at single gene loci. Of particular interest, Asi has been shown to inhibit repair of DNA single-strand breaks, a possible mechanism for its observed comutagenicity and cocarcinogenicity. We propose a cocarcinogenic mode of action in which Asi acts primarily on intermediate cells deficient in cell cycle control at a late stage in a preexisting carcinogenic process. This interaction enhances ge-nomic fragility and accelerates conversion of premalignant lesions to more aggressive, clinically observable tumors. An indirect effect of As, on DNA repair is consistent with the expectation of a nonlinear dose-response rather than the linear dose-response traditionally assumed for mutagenic carcinogens. However, defining the exact nature of this tumor dose-response will require further experimental data on the dose-response for the cellular effects of Asi. Because Asi carcinogenicity is unlikely to be observed in normal experimental animals not exposed to other carcinogens, studies in animals and cell lines deficient in cell cycle control should also be considered. Experimental studies specifically designed to address the key mechanistic and dose-response issues for Asi carcinogenicity are critically needed to support public health policy decisions regarding current environmental exposures to Asi.

Application of a Physiologically Based Pharmacokinetic Model for Reference Dose and Reference Concentration Estimation for Acetone

Recent health risk assessments to propose a Reference Dose (RfD) for acetone (Forsyth, 2001; U.S. EPA, 2001) have been based on the results of an oral subchronic study conducted in rats and mice (Dietz et al., 1991; NTP, 1991). These assessments have utilized the traditional concept of establishing the RfD by determining the lowest experimentally determined No-Observed-Adverse-Effect Level (NOAEL) and applying various Uncertainty Factors (UFs) (U.S. EPA, 1988). This article describes a risk assessment for acetone based on the systemic toxicity observed in subchronic and developmental toxicity studies to estimate an RfD and an inhalation reference concentration (RfC) for acetone. Specifically, this approach examined the subchronic study by Dietz et al. (1991), as well as an inhalation developmental toxicity study on acetone (Mast et al., 1988) and several toxicology studies of isopropanol (IPA). This was accomplished by applying a physiologically based pharmacokinetic (PBPK) model developed previously for IPA and its metabolite acetone (Clewell et al., 2001). The incorporation of the PBPK model into the derivation of an RfD and RfC for acetone allowed for a tissue-based approach rather than an external exposure-based approach, making it possible to derive an oral RfD from an inhalation study. In addition, the use of the PBPK model to analyze data from chronic and reproductive/developmental studies conducted with IPA enabled an assessment of the potential for acetone to produce any of the effects observed in the IPA studies. This analysis provided sufficient information to reduce the need for UFs in the adjustment of the NOAEL from the oral subchronic study for the determination of an RfD. Using the PBPK model in the acetone risk assessment supports a composite UF of 60 for the subchronic study, compared to composite factors of 300 to 3000 in the other recent risk assessments. This difference resulted in an RfD of 16 mg/kg/d, compared to the values of 0.3 to 3 that have previously been estimated (Forsyth, 2001; U.S. EPA, 2001). Considering the results from the inhalation developmental study (Mast et al., 1988) resulted in an RfD of 8.7 mg/kg/d. Using this study also fills a data gap for acetone that exists if only the oral database for acetone is considered for RfD derivation. An RfC of 29ppm was also estimated for acetone using the Mast et al. (1988) study results in combination with the PBPK model. The potential impact of endogenous acetone on a risk assessment for acetone is also discussed.