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Trace-level environmental data typically include values near or below detection and quantitation thresholds where health effects may result from low-concentration exposures to one chemical over time or to multiple chemicals. In a cook stove case study, bias in dibenzo[a,h]anthracene concentration means and standard deviations (SDs) was assessed following censoring at thresholds for selected analysis approaches: substituting threshold/2, maximum likelihood estimation, robust regression on order statistics, Kaplan-Meier, and omitting censored observations. Means and SDs for gas chromatography-mass spectrometry-determined concentrations were calculated after censoring at detection and calibration thresholds, 17% and 55% of the data, respectively. Threshold/2 substitution was the least biased. Measurement values were subsequently simulated from two log-normal distributions at two sample sizes. Means and SDs were calculated for 30%, 50%, and 80% censoring levels and compared to known distribution counterparts. Simulation results illustrated (1) threshold/2 substitution to be inferior to modern after-censoring statistical approaches and (2) all after-censoring approaches to be inferior to including all measurement data in analysis. Additionally, differences in stove-specific group means were tested for uncensored samples and after censoring. Group differences of means tests varied depending on censoring and distributional decisions. Investigators should guard against censoring-related bias from (explicit or implicit) distributional and analysis approach decisions.

PBPK models are well-established frameworks used to describe absorption, distribution, metabolism, and excretion of xenobiotics. To quantify metabolism, a PBPK model for a volatile compound can be calibrated with closed chamber (i.e., vapor uptake) inhalation data. Here, we introduce global optimization as a novel component of the predictive process and use it to illustrate a procedure for metabolic parameter estimation. Male F344 rats were exposed in vapor uptake chambers to initial concentrations of 100, 500, 1000, and 3000 ppm chloroform. Chamber time-course data from these experiments, in combination with optimization using a chemical specific PBPK model, were used to estimate Michaelis-Menten metabolic constants. Matlab® simulation software was used to integrate the mass balance equations and to perform the global optimizations using MEIGO (MEtaheuristics for systems biology and bIoinformatics Global Optimization – Version 64bit, R2016A), a toolbox written for Matlab®). The cost function used the chamber time-course data and least squares to minimize the difference between data and simulation values. The final values estimated for V(max) (maximum metabolic rate) and K(m) (affinity constant) were 1.2 mg/h and a range between 0.0005 and 0.6 mg/L, respectively. Also, cost function plots were used to analyze the dose-dependent capacity to estimate V(max) and K(m) within the experimental range used. Sensitivity analysis was used to assess identifiability for both parameters and show these kinetic data may not be sufficient to identify K(m). The importance of an accurate estimation of metabolism using vapor uptake data is discussed. In summary, this work should help toxicologists interested in optimization techniques understand the overall process employed when calibrating metabolic parameters in a PBPK model with inhalation data.

Trace-level environmental data typically include values near or below detection and quantitation thresholds where health effects may result from low-concentration exposures to one chemical over time or to multiple chemicals. In a cook stove case study, bias in dibenzo[a,h]anthracene concentration means and standard deviations (SDs) was assessed following censoring at thresholds for selected analysis approaches: substituting threshold/2, maximum likelihood estimation, robust regression on order statistics, Kaplan-Meier, and omitting censored observations. Means and SDs for gas chromatography-mass spectrometry-determined concentrations were calculated after censoring at detection and calibration thresholds, 17% and 55% of the data, respectively. Threshold/2 substitution was the least biased. Measurement values were subsequently simulated from two log-normal distributions at two sample sizes. Means and SDs were calculated for 30%, 50%, and 80% censoring levels and compared to known distribution counterparts. Simulation results illustrated (1) threshold/2 substitution to be inferior to modern after-censoring statistical approaches and (2) all after-censoring approaches to be inferior to including all measurement data in analysis. Additionally, differences in stove-specific group means were tested for uncensored samples and after censoring. Group differences of means tests varied depending on censoring and distributional decisions. Investigators should guard against censoring-related bias from (explicit or implicit) distributional and analysis approach decisions.

In a recent U.S. Geological Survey/U.S. Environmental Protection Agency study assessing more than 700 organic compounds in 38 streams, in vitro assays indicated generally low estrogen, androgen, and glucocorticoid receptor activities, with 13 surface waters with 17β-estradiol-equivalent (E2Eq) activities greater than a 1-ng/L estimated effects-based trigger value for estrogenic effects in male fish. Among the 36 samples assayed for mutagenicity in the Salmonella bioassay (reported here), 25% had low mutagenic activity and 75% were not mutagenic. Endocrine and mutagenic activities of the water samples were well correlated with each other and with the total number and cumulative concentrations of detected chemical contaminants. To test the predictive utility of knowledge-base-leveraging approaches, site-specific predicted chemical-gene (pCGA) and predicted analogous pathway-linked (pPLA) association networks identified in the Comparative Toxicogenomics Database were compared with observed endocrine/mutagenic bioactivities. We evaluated pCGA/pPLA patterns among sites by cluster analysis and principal component analysis and grouped the pPLA into broad mode-of-action classes. Measured E2eq and mutagenic activities correlated well with predicted pathways. The pPLA analysis also revealed correlations with signaling, metabolic, and regulatory groups, suggesting that other effects pathways may be associated with chemical contaminants in these waters and indicating the need for broader bioassay coverage to assess potential adverse impacts.

Geologists and archaeologists have long known that the bluestones of Stonehenge came from the Preseli Hills of west Wales, 230km away, but only recently have some of their exact geological sources been identified. Two of these quarries—Carn Goedog and Craig Rhos-y-felin—have now been excavated to reveal evidence of megalith quarrying around 3000 BC—the same period as the first stage of the construction of Stonehenge. The authors present evidence for the extraction of the stone pillars and consider how they were transported, including the possibility that they were erected in a temporary monument close to the quarries, before completing their journey to Stonehenge.

Contaminants of emerging concern (CECs), including per- and polyfluoroalkyl substances (PFAS), are of interest to regulators, water treatment utilities, the general public and scientists. This study measured 17 PFAS in source and treated water from 25 drinking water treatment plants (DWTPs) as part of a broader study of CECs in drinking water across the United States. PFAS were quantitatively detected in all 50 samples, with summed concentrations of the 17 PFAS ranging from, GRAPHICAL ABSTRACT

The discovery of a dismantled stone circle—close to Stonehenge's bluestone quarries in west Wales—raises the possibility that a 900-year-old legend about Stonehenge being built from an earlier stone circle contains a grain of truth. Radiocarbon and OSL dating of Waun Mawn indicate construction c. 3000 BC, shortly before the initial construction of Stonehenge. The identical diameters of Waun Mawn and the enclosing ditch of Stonehenge, and their orientations on the midsummer solstice sunrise, suggest that at least part of the Waun Mawn circle was brought from west Wales to Salisbury Plain. This interpretation complements recent isotope work that supports a hypothesis of migration of both people and animals from Wales to Stonehenge.

BACKGROUND: In developmental and reproductive toxicity studies, analysis of litter-based binary endpoints (e.g., incidence of malformed fetuses) is complex in that littermates often are not entirely independent of one another. It is well established that the litter, not the individual fetus, is the proper independent experimental unit in statistical analysis. Accordingly, analysis is often based on the proportion affected per litter and the litter proportions are analyzed as continuous data. Because these proportional data generally do not meet assumptions of symmetry or normality, data are typically analyzed by nonparametric methods, arcsine square root transformation, or logit transformation. METHODS: We conducted power calculations to compare different approaches (nonparametric, arcsine square root-transformed, logit-transformed, untransformed) for analyzing litter-based proportional data. A reproductive toxicity study with a control and one treated group provided data for two endpoints: prenatal loss, and fertility by in utero insemination (IUI). Type 1 error and power were estimated by 10,000 simulations based on two-sample one-tailed t-tests with varying numbers of litters per group. To further compare the different approaches, we conducted additional analyses with shifted mean proportions to produce illustrative scenarios. RESULTS: Analyses based on logit-transformed proportions had greater power than those based on untransformed or arcsine square root-transformed proportions, or nonparametric procedures. CONCLUSION: The logit transformation is preferred to the other approaches considered when making inferences concerning litter-based proportional endpoints, particularly with skewed distributions. The improved performance of the logit transformation becomes increasingly pronounced as the response proportions are increasingly close to the boundaries of the parameter space.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

A method based on regression modeling was developed to discern the contribution of component chemicals to the toxicity of highly complex, environmentally realistic mixtures of disinfection byproducts (DBPs). Chemical disinfection of drinking water forms DBP mixtures. Because of concerns about possible reproductive and developmental toxicity, a whole mixture (WM) of DBPs produced by chlorination of a water concentrate was administered as drinking water to Sprague–Dawley (S–D) rats in a multigenerational study. Age of puberty acquisition, i.e., preputial separation (PPS) and vaginal opening (VO), was examined in male and female offspring, respectively. When compared to controls, a slight, but statistically significant delay in puberty acquisition was observed in females but not in males. WM-induced differences in the age at puberty acquisition were compared to those reported in S–D rats administered either a defined mixture (DM) of nine regulated DBPs or individual DBPs. Regression models were developed using individual animal data on age at PPS or VO from the DM study. Puberty acquisition data reported in the WM and individual DBP studies were then compared with the DM models. The delay in puberty acquisition observed in the WM-treated female rats could not be distinguished from delays predicted by the DM regression model, suggesting that the nine regulated DBPs in the DM might account for much of the delay observed in the WM. This method is applicable to mixtures of other types of chemicals and other endpoints.

When chemical or microbial contaminants are assessed for potential effect or possible regulation in ambient and drinking waters, a critical first step is determining if the contaminants occur and if they are at concentrations that may cause human or ecological health concerns. To this end, source and treated drinking water samples from29 drinking water treatment plants (DWTPs) were analyzed as part of a two-phase study to determine whether chemical and microbial constituents, many of which are considered contaminants of emerging concern, were detectable in the waters. Of the 84 chemicals monitored in the 9 Phase I DWTPs, 27 were detected at least once in the source water, and 21 were detected at least once in treated drinking water. In Phase II, which was a broader and more comprehensive assessment, 247 chemical and microbial analytes were measured in 25 DWTPs, with 148 detected at least once in the source water, and 121 detected at least once in the treated drinking water. The frequency of detection was often related to the analyte’s contaminant class, as pharmaceuticals and anthropogenic waste indicators tended to be infrequently detected and more easily removed during treatment, while per and polyfluoroalkyl substances and inorganic constituents were both more frequently detected and, overall, more resistant to treatment. The data collected as part of this project will be used to help inform evaluation of unregulated contaminants in surface water, groundwater, and drinking water., GRAPHICAL ABSTRACT

The U.S. Environmental Protection Agency (EPA) is faced with the challenge of efficiently and credibly evaluating chemical safety often with limited or no available toxicity data. The expanding number of chemicals found in commerce and the environment, coupled with time and resource requirements for traditional toxicity testing and exposure characterization, continue to underscore the need for new approaches. In 2005, EPA charted a new course to address this challenge by embracing computational toxicology (CompTox) and investing in the technologies and capabilities to push the field forward. The return on this investment has been demonstrated through results and applications across a range of human and environmental health problems, as well as initial application to regulatory decision-making within programs such as the EPA’s Endocrine Disruptor Screening Program. The CompTox initiative at EPA is more than a decade old. This manuscript presents a blueprint to guide the strategic and operational direction over the next 5 years. The primary goal is to obtain broader acceptance of the CompTox approaches for application to higher tier regulatory decisions, such as chemical assessments. To achieve this goal, the blueprint expands and refines the use of high-throughput and computational modeling approaches to transform the components in chemical risk assessment, while systematically addressing key challenges that have hindered progress. In addition, the blueprint outlines additional investments in cross-cutting efforts to characterize uncertainty and variability, develop software and information technology tools, provide outreach and training, and establish scientific confidence for application to different public health and environmental regulatory decisions.

Iodinated contrast media (ICM) are nonmutagenic agents administered for X-ray imaging of soft tissues. ICM can reach μg/L levels in surface waters because they are administered in high doses, excreted largely unmetabolized, and poorly removed by wastewater treatment. Iodinated disinfection byproducts (I-DBPs) are highly genotoxic and have been reported in disinfected waters containing ICM. We assessed the mutagenicity in Salmonella of extracts of chlorinated source water containing one of four ICM (iopamidol, iopromide, iohexol, and diatrizoate). We quantified 21 regulated and nonregulated DBPs and 11 target I-DBPs and conducted a nontarget, comprehensive broad-screen identification of I-DBPs. We detected one new iodomethane (trichloroiodomethane), three new iodoacids (dichloroiodoacetic acid, chlorodiiodoacetic acid, bromochloroiodoacetic acid), and two new nitrogenous I-DBPs (iodoacetonitrile and chloroiodoacetonitrile). Their formation depended on the presence of iopamidol as the iodine source; identities were confirmed with authentic standards when available. This is the first identification in simulated drinking water of chloroiodoacetonitrile and iodoacetonitrile, the latter of which is highly cytotoxic and genotoxic in mammalian cells. Iopamidol (5 μM) altered the concentrations and relative distribution of several DBP classes, increasing total haloacetonitriles by >10-fold. Chlorination of ICM-containing source water increased I-DBP concentrations but not mutagenicity, indicating that such I-DBPs were either not mutagenic or at concentrations too low to affect mutagenicity.

Prioritizing the risk posed by thousands of chemicals potentially present in the environment requires exposure, toxicity, and toxicokinetic (TK) data, which are often unavailable. Relatively high throughput, in vitro TK (HTTK) assays and in vitro-to-in vivo extrapolation (IVIVE) methods have been developed to predict TK, but most of the in vivo TK data available to benchmark these methods are from pharmaceuticals. Here we report on new, in vivo rat TK experiments for 26 non-pharmaceutical chemicals with environmental relevance. Both intravenous and oral dosing were used to calculate bioavailability. These chemicals, and an additional 19 chemicals (including some pharmaceuticals) from previously published in vivo rat studies, were systematically analyzed to estimate in vivo TK parameters (e.g., volume of distribution [Vd], elimination rate). For each of the chemicals, rat-specific HTTK data were available and key TK predictions were examined: oral bioavailability, clearance, Vd, and uncertainty. For the non-pharmaceutical chemicals, predictions for bioavailability were not effective. While no pharmaceutical was absorbed at less than 10%, the fraction bioavailable for non-pharmaceutical chemicals was as low as 0.3%. Total clearance was generally more underestimated for nonpharmaceuticals and Vd methods calibrated to pharmaceuticals may not be appropriate for other chemicals. However, the steady-state, peak, and time-integrated plasma concentrations of nonpharmaceuticals were predicted with reasonable accuracy. The plasma concentration predictions improved when experimental measurements of bioavailability were incorporated. In summary, HTTK and IVIVE methods are adequately robust to be applied to high throughput in vitro toxicity screening data of environmentally relevant chemicals for prioritizing based on human health risks. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

The discovery of Neolithic houses at Durrington Walls that are contemporary with the main construction phase of Stonehenge raised questions as to their interrelationship. Was Durrington Walls the residence of the builders of Stonehenge? Were the activities there more significant than simply domestic subsistence? Using lipid residue analysis, this paper identifies the preferential use of certain pottery types for the preparation of particular food groups and differential consumption of dairy and meat products between monumental and domestic areas of the site. Supported by the analysis of faunal remains, the results suggest seasonal feasting and perhaps organised culinary unification of a diverse community.

The introduction of drinking water disinfection greatly reduced waterborne diseases. However, the reaction between disinfectants and natural organic matter in the source water leads to an unintended consequence, the formation of drinking water disinfection byproducts (DBPs). The haloace-taldehydes (HALs) are the third largest group by weight of identified DBPs in drinking water. The primary objective of this study was to analyze the occurrence and comparative toxicity of the emerging HAL DBPs. A new HAL DBP, iodoacetaldehyde (IAL) was identified. This study provided the first systematic, quantitative comparison of HAL toxicity in Chinese hamster ovary cells. The rank order of HAL cytotoxicity is tribromoacetaldehyde (TBAL) ≈ chloroacetaldehyde (CAL) > dibromoacetaldehyde (DBAL) ≈ bromochloroacetaldehyde (BCAL) ≈ dibromochloroacetaldehyde (DBCAL) > IAL > bromoacetaldehyde (BAL) ≈ bromodichloroacetaldehyde (BDCAL) > dichloroacetaldehyde (DCAL) > trichloroacetaldehyde (TCAL). The HALs were highly cytotoxic compared to other DBP chemical classes. The rank order of HAL genotoxicity is DBAL > CAL ≈ DBCAL > TBAL ≈ BAL > BDCAL > BCAL ≈ DCAL > IAL. TCAL was not genotoxic. Because of their toxicity and abundance, further research is needed to investigate their mode of action to protect the public health and the environment.

Background Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown. Objective We aimed to evaluate a drinking water mixture of the four regulated THMs and five regulated HAAs in a multigenerational reproductive toxicity bioassay. Methods Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the U.S. Environmental Protection Agency’s maximum contaminant levels (MCLs). Results Maternal water consumption was reduced at ≥ 1,000×; body weights were reduced at 2,000×. Prenatal and postnatal survival were unaffected. F1 pup weights were unaffected at birth but reduced at 2,000× on PND6 and at ≥ 1,000× on PND21. Postweaning F1 body weights were reduced at 2,000×, and water consumption was reduced at ≥ 500×. Males at 2,000× had a small but significantly increased incidence of retained nipples and compromised sperm motility. Onset of puberty was delayed at 1,000× and 2,000×. F1 estrous cycles and fertility were unaffected, and F2 litters showed no effects on pup weight or survival. Histologically, P0 (parental) dams had nephropathy and adrenal cortical pathology at 2,000×. Conclusions A mixture of regulated DBPs at up to 2,000× the MCLs had no adverse effects on fertility, pregnancy maintenance, prenatal survival, postnatal survival, or birth weights. Delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights. Citation Narotsky MG, Klinefelter GR, Goldman JM, DeAngelo AB, Best DS, McDonald A, Strader LF, Murr AS, Suarez JD, George MH, Hunter ES III, Simmons JE. 2015. Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay. Environ Health Perspect 123:564–570; http://dx.doi.org/10.1289/ehp.1408579

The extensification of agricultural systems into marginal lands is a common response to environmental, economic, and political pressures for more cultivable land. Yet the course that extensification takes in particular instances is unpredictable given the choices available to producers. This article investigates an instance of extensification during the late second millennium BCE on the semi-arid Eastern Karak Plateau in west-central Jordan. Architectural, faunal, and archaeobotanical evidence is presented from Khirbat al-Mudayna al-'Aliya, one of several communities that participated in an extensified settlement system on the edge of the Wadi al-Mujib and its tributaries. Producers practiced agriculture and pastoralism in a low-intensity subsistence economy that supported a nucleated settlement of households. Faunal analysis determined goats were kept, and wild animals supplemented diets. Archaeobotanical analysis of charred plant remains from storage bins in a building destroyed by fire indicated that barley was stored in a semi-processed state and that harvesting by uprooting was practiced, thus resulting in the maximization of the straw harvest. The riparian zone beneath the settlement was a key venue for subsistence activities. This Early Iron Age example contrasts with later episodes of extensification whose settlement systems were more dispersed and agro-pastoralist regime more integrated.

Statistical design and environmental relevance are important aspects of studies of chemical mixtures, such as pesticides. We used a dose-additivity model to test experimentally the default assumptions of dose additivity for two mixtures of seven N-methylcarbamates (carbaryl, carbofuran, formetanate, methomyl, methiocarb, oxamyl, and propoxur). The best-fitting models were selected for the single-chemical dose-response data and used to develop a combined prediction model, which was then compared with the experimental mixture data. We evaluated behavioral (motor activity) and cholinesterase (ChE)-inhibitory (brain, red blood cells) outcomes at the time of peak acute effects following oral gavage in adult and preweanling (17 days old) Long-Evans male rats. The mixtures varied only in their mixing ratios. In the relative potency mixture, proportions of each carbamate were set at equitoxic component doses. A California environmental mixture was based on the 2005 sales of each carbamate in California. In adult rats, the relative potency mixture showed dose additivity for red blood cell ChE and motor activity, and brain ChE inhibition showed a modest greater-than additive (synergistic) response, but only at a middle dose. In rat pups, the relative potency mixture was either dose-additive (brain ChE inhibition, motor activity) or slightly less-than additive (red blood cell ChE inhibition). On the other hand, at both ages, the environmental mixture showed greater-than additive responses on all three endpoints, with significant deviations from predicted at most to all doses tested. Thus, we observed different interactive properties for different mixing ratios of these chemicals. These approaches for studying pesticide mixtures can improve evaluations of potential toxicity under varying experimental conditions that may mimic human exposures.

In rodent bioassays where chemicals are administered in the drinking water, water consumption data for individual animals are needed to estimate chemical exposures accurately. If multiple animals share a common water source, as occurs in some studies, only the total amount of drinking water consumed by all animals utilizing the common source is directly measurable, and water consumption rates for individual animals are not available. In the Four Lab Study of the US Environmental Protection Agency, which included a multigenerational rodent bioassay, a complex mixture of drinking water disinfection by-products was delivered to multiple Sprague-Dawley rats from a common drinking water container. To estimate disinfection by-product mixture exposure for each animal, authors developed four log-linear regression models to allocate water consumption among rats sharing a common water container. The four models represented three animal lifestages: Gestation, Lactation, and Postweaning, with separate Postweaning models for male and female. Authors used data from six Sprague-Dawley rat bioassays to develop these models from available individual cage data for the Postweaning models, and available individual animal data for the Gestation and Lactation models. The r(2) values for the model fits were good, ranging from 0.67 to 0.92. The Gestation and Lactation models were generally quite accurate in predicting average daily water consumption whereas the Postweaning models were less robust. These models can be generalized for use in other reproductive and developmental bioassays where common water sources are used and data on the explanatory variables are available.

In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA’s Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90%) to detect pup weight decreases, while providing the most power to detect increased prenatal loss.

Fixed-ratio ray designs have been used for detecting and characterizing interactions of large numbers of chemicals in combination. Single-chemical dose-response data are used to predict an “additivity curve” along an environmentally relevant ray. A “mixture curve” is estimated from the mixture dose-response data along the ray. A test of additivity is equivalent to a test of coincidence of these two curves, which is based on the traditional hypothesis testing framework that assumes additivity in the null hypothesis and rejects with evidence of interaction. However, failure to reject may be due to lack of statistical power, making the claim of additivity problematic. As a solution we have developed rigorous methodology to test for additivity using statistical equivalence testing logic in which additivity is claimed based on pre-specified biologically important additivity margins, if the data support such a claim. Using the principle of confidence interval inclusion, a confidence region about the difference of meaningful functions of model parameters from the mixture model and that predicted under additivity is computed. When the confidence region is completely contained within the additivity margins then additivity is claimed with a Type I error rate chosen a priori to be some acceptably small value. The method is illustrated using an environmentally relevant fixed-ratio mixture of nine haloacetic acids where cytotoxic response is measured.

As part of the DOE RIA R&D effort we investigated the possibilities and problems of beam strippers in the different heavy ion accelerator components of a possible Rare Isotope Accelerator (RIA) facility. We focused on two beam stripping positions in the RIA heavy ion driver where benchmark currents of up to 5 particle μA 238-U were projected at energies of 10.5 MeV/u and 85 MeV/u respectively. In order to select feasible stripper materials, data from experiments with Uranium beams at Texas A&M and GSI were evaluated. Based on these results thermal estimates for a possible design were calculated and cooling simulations with commercially available software performed. Additionally, we performed simulations with the GEANT4 code on evaluating the radiation environment for our beam stripping solution at the 85 MeV/u position in the RIA driver.

When an interaction has been detected among the chemicals in a mixture, it may be of interest to predict the interaction threshold. A method is presented for estimation of an interaction threshold along a mixture ray which allows differences in the shapes of the dose-response curves of the individual components (e.g., mixtures of full and partial agonists with differing response maxima). A point estimate and confidence interval for the interaction threshold may be estimated. The methods are illustrated with data from a study of a mixture of 18 polyhalogenated aromatic hydrocarbons (PHAHs) in rats exposed by oral gavage for four consecutive days. Serum total thyroxine (T4) was the response variable. Previous analysis of these data demonstrated a dose-dependent interaction among the 18 chemicals in the mixture, with additivity suggested in the lower portion of the dose-response curve and synergy (greater than additive response) in the higher portion of the dose-response curve. The present work builds on this analysis by construction of an interaction threshold model along the mixture ray. This interaction threshold model has two components: an implicit additivity region and an explicit region that describes the departure from additivity; the interaction threshold is the boundary between the two regions. Estimation of the interaction threshold within the observed experimental region suggested evidence of additivity in the low dose region. Total doses of the mixture that exceed the upper limit of the confidence interval on the interaction threshold were associated with a greater-than-additive interaction.

The estimation of risk following exposure to mixtures is an important feature of pesticide risk assessment. Also of concern is the potential for increased sensitivity of the young to pesticide toxicity. We have conducted interaction studies using a mixture of five organophosphorus (OP) pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion) in both adult (published previously) and preweanling rats using a fixed-ratio ray design. In the present study, cholinesterase inhibition and behavioral changes (motor activity, gait, and tail-pinch response) were measured in 17-day-old Long-Evans male rats following acute exposure to the OPs. The ratio of pesticides in the mixture reflected the relative dietary exposure estimates projected by the U.S. Environmental Protection Agency Dietary Exposure Evaluation Model. Dose-response data were collected for each OP alone, which were used (alone or in conjunction with the mixture data) to build an additivity model to predict the effects of the pesticide mixture along a ray of increasing total doses, using the same fixed ratio of components. The mixture data (full ray) were similarly modeled and statistically compared to the additivity model along the ray. Since malathion has been shown to produce synergistic interactions with certain OPs, it was of interest to evaluate the influence of malathion in this study. A second pesticide mixture, without malathion (reduced ray), was tested using the same dose levels of the remaining four OPs. Analysis of the full ray revealed significant greater-than-additive responses for all endpoints. The magnitude of this shift ranged from two- to threefold for estimates of the ED(20) and ED(50). The deviation from additivity was also detected in the reduced ray for all but two endpoints (motor activity and tail-pinch response); however, for all endpoints, the reduced ray was significantly different from the full ray. Thus, greater-than-additive responses were detected in preweanling rats with this OP mixture, and this effect can only partially be attributed to the malathion in the mixture.

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and poly-brominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose–response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

The relationship between the concentration of trichloroethylene (TCE) in the brain and changes in brain function, indicated by the amplitude of steady-state pattern-elicited visual evoked potentials (VEP), was evaluated in Long-Evans rats. VEPs were recorded from visual cortex following stimulation of the eyes and, thus, reflect the function of the afferent visual pathway and, in broad terms, may be indicative of overall brain function. The concentration of TCE in the brain at the time of VEP testing (i.e., momentary brain concentration) was hypothesized to predict the amplitude of the VEP across a range of inhalation concentrations, both during and after exposure. Awake restrained rats were exposed to clean air or TCE in the following combinations of concentration and duration: 500 ppm (4 h), 1000 ppm (4 h), 2000 (2 h), 3000 ppm (1.3 h), 4000 ppm (1 h), and 5000 ppm (0.8 h). VEPs were recorded several times during the exposure session, and afterward for experimental sessions of less than 4 h total duration (i.e., concentrations from 2000 to 5000 ppm). The sample collection time for each VEP was about 1 min. Brain concentrations of TCE were predicted using a physiologically based pharmacokinetic (PBPK) model. VEP waveforms were submitted to spectral analysis, and the amplitude of the largest response component, occurring at twice the temporal stimulation rate (F2), was measured. Exposure to all air concentrations of TCE in the study reduced F2 amplitude. The reduction of F2 amplitude was proportional to momentary brain TCE concentration during and after exposure. A logistical function fit to the combined data from all exposure conditions described a statistically significant relationship with 95% confidence limits between brain TCE concentration and F2 amplitude. The results support the hypothesis that momentary brain concentration of TCE is an appropriate dose metric to describe the effects of acute TCE inhalation exposure on rat VEPs. The combination of the PBPK model predicting brain TCE concentration from the exposure conditions with the logistical function predicting F2 amplitude from the brain TCE concentration constitute a quantitative exposure-dose-response model describing an acute change in neurological function following exposure to an important hazardous air pollutant.

We are developing an exposure–dose–response (EDR) model for volatile organic compounds (VOCs) to predict acute effects of VOCs on nervous system function from exposure data (concentration and duration of inhalation). This model contains both toxicokinetic and toxicodynamic components. One advantage of the EDR model will be its ability to relate in vitro effects of solvents on cellular ion channels (putative targets) to in vivo effects, using a combination of physiologically-based toxicokinetic (PBTK) modeling (to estimate VOC concentrations in the blood and brain) and in vitro studies to clarify the mode of action of the VOCs. Recent work in vitro has focused on quantifying the inhibitory effects of toluene, trichloroethylene (TCE) and perchloroethylene (PERC) on ion channel currents. All three VOCs inhibit current through voltage-sensitive calcium channels (VSCCs) in pheochromocytoma cells; PERC blocked calcium currents and altered the current–voltage relationship at lower concentrations than did toluene or TCE. Recombinant nicotinic acetylcholine receptors (nAChRs), expressed in Xenopus oocytes, were also inhibited by PERC and toluene in a concentration-dependent manner. PERC inhibited α7 receptors more than α4β2 receptors in recombinant human and rat nAChRs. However, human and rat α7 receptors were equally sensitive to PERC and TOL. These in vitro studies will be used to identify an appropriate neuronal receptor system to serve as an index of acute effects of VOCs in vivo. The PBTK model incorporates physiological input parameters derived from radiotelemetered heart rate data from rats performing operant tests of cognitive and motor functions. These studies should improve predictions of target organ concentrations of inhaled VOCs in subjects actively performing behavioral tests over a range of physical activity levels.

Risk assessment procedures can be improved through better understanding and use of tissue dose information and linking tissue dose level to adverse outcomes. For volatile organic compounds, such as toluene and trichloroethylene (TCE), blood and brain concentrations can be estimated with physiologically based pharmacokinetic (PBPK) models. Acute changes in the function of the nervous system can be linked to the concentration of test compounds in the blood or brain at the time of neurological assessment. This set of information enables application to a number of risk assessment situations. For example, we have used this approach to recommend duration adjustments for acute exposure guideline levels (AEGLs) for TCE such that the exposure limits for each exposure duration yield identical tissue concentrations at the end of the exposure period. We have also used information on tissue concentration at the time of assessment to compare sensitivity across species, adjusting for species-specific pharmacokinetic differences. Finally this approach has enabled us to compare the relative sensitivity of different compounds on a tissue dose basis, leading to expression of acute solvent effects as ethanol-dose equivalents for purposes of estimating cost–benefit relationships of various environmental control options.

This article presents and illustrates an approach to designing and analyzing studies involving mixtures/combinations of drugs or chemicals along fixed-ratio rays of the drugs or chemicals for generalized linear models. When interest can be restricted to a specific ray, we consider fixed-ratio ray designs to reduce the amount of experimental effort. When a ray design is used, we have shown that the hypothesis of additivity can be rejected when higher order polynomial terms are required in the total dose-response model. Thus, it is important that we have precise parameter estimates for these higher order polynomial terms in the linear predictor. We have developed methodology for finding a Ds-optimal design based on this subset of the terms in the linear predictor.

An important environmental and regulatory issue is the protection of human health from potential adverse effects of cumulative exposure to multiple chemicals. Earlier literature suggested restricting inference to specific fixed-ratio rays of interest. Based on appropriate definitions of additivity, single chemical data are used to predict the relationship among the chemicals under the zero-interaction case. Parametric comparisons between the additivity model and the model fit along the fixed-ratio ray(s) are used to detect departure from additivity. Collection of data along reduced fixed-ratio rays, where subsets of chemicals of interest are removed from the mixture and the remaining compounds are at the same relative ratios as considered in the full ray, allow researchers to make inference about the effect of the removed chemicals. Methods for fitting simultaneous confidence bands about the difference between the best fitting model and the model predicted under additivity are developed to identify regions along the rays where significant interactions occur. This general approach is termed the “single chemicals required” (SCR) method of analysis. A second approach, termed “single chemicals not required” (SCNR) method of analysis, is based on underlying assumptions about the parameterization of the response surface. Under general assumptions, polynomial terms for models fit along fixed-ratio rays are associated with interaction terms. Consideration is given to the case where only data along the mixture rays are available. Tests of hypotheses, which consider interactions due to subsets of chemicals, are also developed.

Risk assessments often must consider exposures that vary over time or for which the exposure duration of concern differs from the available data, and a variety of extrapolation procedures have been devised accordingly. The present experiments explore the relationship(s) between exposure concentration (C) and time (t) to investigate procedures for assessing the risks of short-term solvent exposures. The first hypothesis tested was that the product of C x t would produce a constant health effect (Haber's rule). The second hypothesis tested was that exposure conditions produce effects in proportion to the tissue concentrations created. Awake, adult, male Long-Evans (LE) rats were exposed to trichloroethylene (TCE) vapor in a head-only exposure chamber while pattern onset/offset visual evoked potentials (VEPs) were recorded. Exposure conditions were designed to provide C x t products of 0 ppm/h (0 ppm for 4 h) or 4000 ppm/h created through four exposure scenarios: 1000 ppm for 4 h; 2000 ppm for 2 h; 3000 ppm for 1.3 h; or 4000 ppm for 1h (n = 9-10/concentration). The amplitude of the VEP frequency double component (F2) was decreased significantly by exposure; this decrease was related to C but not to t or to the C x t product, indicating that Haber's rule did not hold. The mean amplitude (+/- SEM in muV) of the F2 component in the control and treatment groups measured 4.4 +/- 0.5 (0 ppm/4 h), 3.1 +/- 0.5 (1000 ppm/4 h), 3.1 +/- 0.4 (2000 ppm/2 h), 2.3 +/- 0.3 (3000 ppm/1.3 h), and 1.9 +/- 0.4 (4000 ppm/1 h). A physiologically based pharmacokinetic (PBPK) model was used to estimate the concentrations of TCE in the brain achieved during each exposure condition. The F2 amplitude of the VEP decreased monotonically as a function of the estimated peak brain concentration but was not related to the area under the curve (AUC) of the brain TCE concentration. In comparison to estimates from the PBPK model, extrapolations based on Haber's rule yielded approximately a 6-fold error in estimated exposure duration when extrapolating across only a 4-fold change in exposure concentration. These results indicate that the use of a linear form of Haber's rule will not predict accurately the risks of acute exposure to TCE, nor will an estimate of AUC of brain TCE. However, an estimate of the brain TCE concentration at the time of VEP testing predicted the effects of TCE across exposure concentrations and durations.

Positive epidemiologic and toxicologic studies underline the need for assessment of human health risks from ubiquitous exposures to mixtures of disinfection by-products (DBPs). Typical human exposures to DBPs are at extremely low levels; thus, health risk is likely to be attributable to multiple, rather than single, DBP exposures. This article suggests three approaches to evaluating health risks from DBP mixtures: (1) toxicologic studies of simple defined mixtures, (2) toxicologic studies using reproducible disinfection scenario samples, and (3) toxicologic or epidemiologic studies conducted directly on drinking water samples. The authors believe all three approaches are valid and can provide data applicable to DBP risk assessment. They also postulate that single-chemical toxicologic studies are, by themselves, insufficient to fully characterize DBP health risks. A concentrated effort by a multidisciplinary team of researchers is necessary to generate appropriate DBP mixtures health data.

Chemical disinfection of water is a major public health triumph of the 20th century. Dramatic decreases in both morbidity and mortality of waterborne diseases are a direct result of water disinfection. With these important public health benefits comes low-level, chronic exposure to a very large number of disinfection byproducts (DBPs), chemicals formed through reaction of the chemical disinfectant with naturally occurring inorganic and organic material in the source water. This article provides an overview of joint research planning by scientists residing within the various organizations of the U.S. Environmental Protection Agency Office of Research and Development. The purpose is to address concerns related to potential health effects from exposure to DBPs that cannot be addressed directly from toxicological studies of individual DBPs or simple DBP mixtures. Two factors motivate the need for such an investigation of complex mixtures of DBPs: a) a significant amount of the material that makes up the total organic halide and total organic carbon portions of the DBPs has not been identified; and b) epidemiologic data, although not conclusive, are suggestive of potential developmental, reproductive, or carcinogenic health effects in humans exposed to DBPs. The plan is being developed and the experiments necessary to determine the feasibility of its implementation are being conducted by scientists from the National Health and Environmental Effects Research Laboratory, the National Risk Management Research Laboratory, the National Exposure Research Laboratory, and the National Center for Environmental Assessment.

Strategies are needed for assessing the risks of exposures to airborne toxicants that vary over concentrations and durations. The goal of this project was to describe the relationship between the concentration and duration of exposure to inhaled trichloroethylene (TCE), a representative volatile organic chemical, tissue dose as predicted by a physiologically based pharmacokinetic model, and neurotoxicity. Three measures of neurotoxicity were studied: hearing loss, signal detection behavior, and visual function. The null hypothesis was that exposure scenarios having an equivalent product of concentration and duration would produce equal toxic effects, according to the classic linear form of Haber's Rule ((italic)C(/italic) times t = k), where C represents the concentration, t, the time (duration) of exposure, and k, a constant toxic effect. All experiments used adult male, Long-Evans rats. Acute and repeated exposure to TCE increased hearing thresholds, and acute exposure to TCE impaired signal detection behavior and visual function. Examination of all three measures of neurotoxicity showed that if Haber's Rule were used to predict outcomes across exposure durations, the risk would be overestimated when extrapolating from shorter to longer duration exposures, and underestimated when extrapolating from longer to shorter duration exposures. For the acute effects of TCE on behavior and visual function, the estimated concentration of TCE in blood at the time of testing correlated well with outcomes, whereas cumulative exposure, measured as the area under the blood TCE concentration curve, did not. We conclude that models incorporating dosimetry can account for differing exposure scenarios and will therefore improve risk assessments over models considering only parameters of external exposure.

Recent efforts to update cumulative risk assessment procedures to incorporate nonchemical stressors ranging from physical to psychosocial reflect increased interest in consideration of the totality of variables affecting human health and the growing desire to develop community-based risk assessment methods. A key roadblock is the uncertainty as to how nonchemical stressors behave in relationship to chemical stressors. Physical stressors offer a reasonable starting place for measuring the effects of nonchemical stressors and their modulation of chemical effects (and vice versa), as they clearly differ from chemical stressors; and "doses" of many physical stressors are more easily quantifiable than those of psychosocial stressors. There is a commonly held belief that virtually nothing is known about the impact of nonchemical stressors on chemically mediated toxicity or the joint impact of coexposure to chemical and nonchemical stressors. Although this is generally true, there are several instances where a substantial body of evidence exists. A workshop titled "Cumulative Risk: Toxicity and Interactions of Physical and Chemical Stressors" held at the 2013 Society of Toxicology Annual Meeting provided a forum for discussion of research addressing the toxicity of physical stressors and what is known about their interactions with chemical stressors, both in terms of exposure and effects. Physical stressors including sunlight, heat, radiation, infectious disease, and noise were discussed in reference to identifying pathways of interaction with chemical stressors, data gaps, and suggestions for future incorporation into cumulative risk assessments.

As part of a multidisciplinary health effects study, the nephrotoxicity of complex industrial waste mixtures was assessed. Adult, male Fischer 344 rats were gavaged with samples of complex industrial waste and nephrotoxicity evaluated 24 hr later. Of the 10 tested samples, 4 produced increased absolute or relative kidney weight, or both, coupled with a statistically significant alteration in at least one of the measured serum parameters (urea nitrogen (BUN), creatinine (CREAT), and BUN/CREAT ratio). Although the waste samples had been analyzed for a number of organic chemicals and 7 of the 10 samples were analyzed also for 12 elemental metals and metalloids, their nephrotoxicity was not readily predicted from the partial chemical characterization data. Because the chemical form or speciation of the metals was unknown, it was not possible to estimate their contribution to the observed biological response. Various experimental approaches, including use of real-world complex mixtures, chemically defined synthetic mixtures, and simple mixtures, will be necessary to adequately determine the potential human health risk from exposure to complex chemical mixtures.

It is predicted that climate change has increased the frequency and intensity of natural disturbances and weather related natural disasters. Rural forest-based communities are especially vulnerable to changes in natural disturbance regimes influenced by climate change because their economic, social, and cultural aspects of life are closely linked to the local environment and climate. In this article, we discuss the impacts of wildfires on communities as an example of how natural disturbances impact humans. The impacts to humans of wildfire is indicative of the type of effects that other natural disturbances such as widespread insect infestation, landslides, floods, drought, storms, avalanches, permafrost melt, forest diseases, erosion, and gradual ecosystem change can have on communities. First Nations communities may be significantly and uniquely impacted by natural disturbances and climate change due to their remote location, strong connection, and heavy reliance on the environment for subsistence and in preserving their culture and their unique and often vulnerable economic situation. We describe the uncertainty of predicting the frequency and intensity of natural disturbances in a particular location. We suggest that the most effective management response to address this uncertainty is to focus on reducing vulnerability and increasing community resilience. Finally, we list some of the management strategies and tools that communities and those that work with them have been using in British Columbia and elsewhere to increase community resilience to natural disturbances and climate change.

The role of nonchemical stressors in modulating the human health risk associated with chemical exposures is an area of increasing attention. On 9 March 2011, a workshop titled “Approaches for Incorporating Nonchemical Stressors into Cumulative Risk Assessment” took place during the 50th Anniversary Annual Society of Toxicology Meeting in Washington D.C. Objectives of the workshop included describing the current state of the science from various perspectives (i.e., regulatory, exposure, modeling, and risk assessment) and presenting expert opinions on currently available methods for incorporating nonchemical stressors into cumulative risk assessments. Herein, distinct frameworks for characterizing exposure to, joint effects of, and risk associated with chemical and nonchemical stressors are discussed.

A summary of the 2010 International Indigenous Earth conference in Penticton British Columbia and the current status of the forthcoming conference proceedings.

Reactions between chemicals used to disinfect drinking water and compounds present in source waters produce chemical mixtures containing hundreds of disinfection byproducts (DBPs). Although the results have been somewhat inconsistent, some epidemiological studies suggest associations may exist between DBP exposures and adverse developmental outcomes. The potencies of individual DBPs in rodent and rabbit developmental bioassays suggest that no individual DBP can account for the relative risk estimates reported in the positive epidemiologic studies, leading to the hypothesis that these outcomes could result from the toxicity of DBP mixtures. As a first step in a mixtures risk assessment for DBP developmental effects, this paper identifies developmentally toxic DBPs and examines data relevant to the mode of action (MOA) for DBP developmental toxicity. We identified 24 developmentally toxic DBPs and four adverse developmental outcomes associated with human DBP exposures: spontaneous abortion, cardiovascular defects, neural tube defects, and low birth weight infancy. A plausible MOA, involving hormonal disruption of pregnancy, is delineated for spontaneous abortion, which some epidemiologic studies associate with total trihalomethane and bromodichloromethane exposures. The DBP data for the other three outcomes were inadequate to define key MOA steps.