A simulation study of extrapolation uncertainty in exposure assessment - Use of pilot study results for site investigation.

Accurate characterization of soil contaminant concentrations is often crucial for assessing risks to human and ecological health. However, fine-scale assessments of large tracts of land can be cost prohibitive due to the number of samples needed. One solution to this problem is to extrapolate sampling results from one area to another unsampled area. In the absence of a validated extrapolation methodology, regulatory agencies have employed policy-based techniques for large sites, but the likelihood of decision errors resulting from these extrapolations is largely unexplored. This study describes the results of a simulation study aimed at guiding environmental sampling for sites where extrapolation concepts are of interest. The objective of this study is to provide practical recommendations to regulatory agencies for extrapolating sampling results on large tracts of land while minimizing errors that are detrimental to human health. A variety of site investigation scenarios representative of environmental conditions and sampling schemes were tested using adaptive sampling when collecting discrete samples or applying incremental sampling methodology (ISM). These simulations address extrapolation uncertainty in cases where a Pilot Study might result in either false noncompliance or false compliance conclusions. A wide range of plausible scenarios were used that reflect the variety of heterogeneity seen at large sites. This simulation study demonstrates that ISM can be reliably applied in a Pilot Study for purposes of extrapolating the outcome to a large area site because it decreases the likelihood of false non-compliance errors while also providing reliable estimates of true compliance across unsampled areas. The results demonstrate how errors depend on the magnitude of the 95% upper confidence limit for the mean concentration (95UCL) relative to the applicable action level, and that error rates are highest when the 95UCL is within 10%-40% of the action level. The false compliance rate can be reduced to less than 5% when 30% or more of the site is characterized with ISM. False compliance error rates using ISM are insensitive to the fraction of the decision units (DUs) that are characterized with three replicates (with a minimum of 10 percent), so long as 95UCLs are calculated for the DUs with one replicate using the average coefficient of variation from the three replicate DUs.

High-precision Pb isotopes of drinking water lead pipes: Implications for human exposure to industrial Pb in the United States.

Millions of lead (Pb) pipes are still used in the drinking water distribution systems in many regions in the world. Human exposure to Pb from contaminated drinking water continues to be of concern in the United States (U.S.), as illustrated by the widely publicized "Flint Water Crisis" in 2015. The Pb isotopic composition of Pb-pipes potentially can be useful to identify human exposure to Pb from lead service lines (LSLs). In addition, as the LSLs were likely manufactured from similar industrial Pb sources as other Pb objects and materials in the USA, the Pb-pipes isotope data can provide information about the overall isotopic composition of the U.S. industrial Pb. In this work we present high-precision Pb isotope data from Pb-pipes excavated from different U.S. municipalities. The Pb-pipes show an extremely wide range of Pb isotopic compositions, with 206Pb/204Pb ranging from 17.004 to 22.010, 207Pb/204Pb from 15.460 to 15.921, and 208Pb/204Pb from 36.687 to 41.120. The wide isotope range is observed even in a single town, suggesting that no regional Pb isotope patterns can be expected within the continental USA. However, the high-precision MC-ICP-MS Pb data form a clear linear trend that, depending on the context, can be used to identify human Pb exposure. Furthermore, as the linear trend is a result of utilization of Pb ores from different domestic and international sources and secondary recycling of metallic Pb, it is likely representative of the overall isotopic composition of the U.S. industrial Pb pool. Therefore, the identified trend is the most accurate isotope representation of the U.S. anthropogenic Pb at present and can be used as first-order evaluation to determine if a person with elevated blood Pb levels was exposed to U.S. industrial Pb sources.

Effect of soil particle size and extraction method on the oral bioaccessibility of arsenic.

Recent findings indicate that incidental ingestion of soil by humans primarily involves soil particles <150 µm, rather than <250 µm-sized fraction previously used for most oral bioaccessibility and bioavailability studies. It was postulated that a greater soil surface area in the finer fraction (<150 versus <250 µm) might increase oral bioaccessibility of arsenic (As) in soil. Bioaccessibility and concentrations of As were compared in <150 and <250 µm fractions of 18 soil samples from a variety of arsenic-contaminated sites. The two methods used to measure bioaccessibility were compared - EPA Method 1340 and the California Arsenic Bioaccessibility (CAB) method. Arsenic concentrations were nearly the same or higher in the <150 fraction compared with <250 µm. EPA Method 1340 and the CAB method presented significantly different bioaccessibility results, as well as estimated relative oral bioavailability (RBA) based upon algorithms specific to the methods, but there was no marked difference for <150 and <250 µm soil fractions within either method. When compared with RBA determined previously for these soil samples in vivo in non-human primates, EPA Method 1340 was generally more predictive than the CAB method. Data suggest that soil- or site-specific factors control bioaccessibility under either method and that the test method selected is more important than the particle size fraction (<150 or <250) in using these in vitro methods to predict As RBA for use in risk assessment.

Review of regulatory reference values and background levels for heavy metals in the human diet.

The U.S. Food and Drug Administration (US FDA) has identified dietary exposure to heavy metals as a public health concern, focusing particularly on arsenic, cadmium, lead, and mercury. One way to determine current risk is to compare established safe exposure limits (reference values) with current population-based dietary background levels. Information on reference values and dietary background exposures for these metals and chromium were critically evaluated in support of an interactive risk assessment screening tool (Heavy Meals Screening Tool [HMST]). Cadmium, arsenic, and mercury background exposures from food and water were found to be below current safe US regulatory limits based on non-cancer effects, while lead background exposures were nearly equivalent to the US FDA's newest interim reference level for children. Because detections of chromium in foods are infrequent and data on speciation (trivalent versus hexavalent) are limited, chromium was excluded from the HMST. The focus of this work was to present U.S. based reference and background exposure values, although the tool can use inputs that may be more appropriate for other countries, cultures, and situations. With emerging science, new health endpoints, and changes in food consumption trends, both reference values and background exposure levels are likely to evolve.

Plasma l-indospicine and 3-nitropropionic acid in ponies fed creeping indigo: Comparison with results from an episode of presumptive creeping indigo toxicosis.

BACKGROUND: Creeping indigo (Indigofera spicata) toxicosis is an emerging problem among horses in Florida and bordering states. OBJECTIVES: To quantify the putative toxins l-indospicine (IND) and 3-nitropropionic acid (NPA) in creeping indigo collected from multiple sites and to measure plasma toxin concentrations in ponies fed creeping indigo and horses with presumptive creeping indigo toxicosis. STUDY DESIGN: Experimental descriptive study with descriptive observational field investigation. METHODS: Air-dried creeping indigo was assayed for IND and NPA content. Five ponies were fed chopped creeping indigo containing 1 mg/kg/day of IND and trace amounts of NPA for 5 days, then observed for 28 days. Blood samples from these ponies and from horses involved in a presumptive creeping indigo toxicosis were assayed for IND and NPA. RESULTS: IND in creeping indigo plants was 0.4-3.5 mg/g dry matter whereas NPA was <0.01 to 0.03 mg/g. During creeping indigo feeding, clinical and laboratory signs were unchanged except for significant weight loss (median 6%, range 2%-9%; p = .04) and significant increase from baseline plasma protein concentration (median 16 g/L, range 8-25 g/L; p < .001). These changes could not definitively be ascribed to creeping indigo ingestion. Plasma IND rose to 3.9 ± 0.52 mg/L on day 6. Pharmacokinetic modelling indicated an elimination half-life of 25 days and a steady state plasma concentration of 22 mg/L. Plasma IND concentration in sick horses during an incident of creeping indigo toxicosis was approximately twice that of clinically normal pasture mates. Plasma NPA was <0.05 mg/L in all samples. MAIN LIMITATIONS: Creeping indigo used in the feeding trial may not be representative of plants involved in creeping indigo toxicosis. There was no control group without creeping indigo in the feeding trial. CONCLUSIONS: Indospicine can be detected in blood of horses consuming creeping indigo and the toxin accumulates in tissues and clears slowly. The role of NPA in the neurological signs of this syndrome is unclear.

Per- and Polyfluoroalkyl Substance Toxicity and Human Health Review: Current State of Knowledge and Strategies for Informing Future Research.

Reports of environmental and human health impacts of per- and polyfluoroalkyl substances (PFAS) have greatly increased in the peer-reviewed literature. The goals of the present review are to assess the state of the science regarding toxicological effects of PFAS and to develop strategies for advancing knowledge on the health effects of this large family of chemicals. Currently, much of the toxicity data available for PFAS are for a handful of chemicals, primarily legacy PFAS such as perfluorooctanoic acid and perfluorooctane sulfonate. Epidemiological studies have revealed associations between exposure to specific PFAS and a variety of health effects, including altered immune and thyroid function, liver disease, lipid and insulin dysregulation, kidney disease, adverse reproductive and developmental outcomes, and cancer. Concordance with experimental animal data exists for many of these effects. However, information on modes of action and adverse outcome pathways must be expanded, and profound differences in PFAS toxicokinetic properties must be considered in understanding differences in responses between the sexes and among species and life stages. With many health effects noted for a relatively few example compounds and hundreds of other PFAS in commerce lacking toxicity data, more contemporary and high-throughput approaches such as read-across, molecular dynamics, and protein modeling are proposed to accelerate the development of toxicity information on emerging and legacy PFAS, individually and as mixtures. In addition, an appropriate degree of precaution, given what is already known from the PFAS examples noted, may be needed to protect human health. Environ Toxicol Chem 2021;40:606-630. © 2020 SETAC.

Dose-response assessment for impaired memory from chronic exposure to domoic acid among native American consumers of razor clams.

Domoic acid (DA) is a marine neurotoxin that accumulates in filtering shellfish during harmful algal blooms. A health protection limit of 20 ppm DA in razor clams (RC) has been set based principally upon an episode of acute DA toxicity in humans that included Amnesic Shellfish Poisoning among survivors. The objective of this study was to determine the dose-response relationship between estimated DA exposure through RC consumption and memory loss in Washington state Native Americans from 2005 to 2015. Results from total learning recall (TLR) memory scores were compared before and after the highest DA exposures. A decrease in TLR was related to DA dose (p < 0.01) regardless whether the effect was assumed to be transient or lasting, and whether the dose was expressed as an average daily dose or an average dose per meal. Benchmark dose modeling identified BMDL10 values of 167 ng/kg-day and 2740 ng/kg-meal assuming a transient effect, and 196 ng/kg-day and 2980 ng/kg-meal assuming no recovery of function occurs. These DA dose thresholds for a measurable memory function reduction observed in this study of clam consumers are well below the safe acute dose underpinning the current regulatory DA limit of 20 ppm (ca. 60 µg/kg).

Probabilistic risk assessment for high-end consumers of seafood on the northeastern Gulf coast.

The Deepwater Horizon oil spill (April 20, 2010) caused concern regarding Gulf seafood safety. Communities were skeptical of governmental risk assessments because they did not take into account the higher consumption of seafood along coastal areas. The objective of this study was to perform a probabilistic risk assessment based on the consumption rates of high-end consumers of Gulf seafood. We utilized seafood consumption data from five communities across the northeastern Gulf of Mexico. This study collected finfish, shrimp, blue crab, and oysters from these communities and analyzed their tissues for polynuclear aromatic hydrocarbons (PAHs). A probabilistic risk assessment was performed using population-specific seafood consumption rates and body weights for commercial fishers, recreational fishers, and a Filipino-American community. For non-cancer effects, 95th percentile hazard quotients for these targeted populations ranged between 1.84E-04 to 5.39E-03 for individual seafood types. The 95th percentile hazard indices for total seafood consumption ranged from 3.45E-03 to 8.41E-03. Based on total seafood consumption, highest hazard indices were modeled for the Filipino-American community followed by commercial and recreational fishers. Despite higher consumption rates, hazard indices for the high-end consumers targeted in this study were two to three orders of magnitude below the regulatory limit of 1.

Derivation of a chronic reference dose for perfluorohexane sulfonate (PFHxS) for reproductive toxicity in mice.

Perfluorohexane sulfonate (PFHxS) is a six-carbon perfluoroalkyl sulfonic acid that was used as an industrial surfactant, but is now found as an environmental contaminant worldwide. In addition to its use as an industrial surfactant, it is a legacy contaminant from the use of aqueous film-forming foams. Despite its widespread occurrence in the environment and evidence of biological activity associated with PFHxS and similar perfluoroalkyl sulfonic acids in rodents, there is no oral toxicity value currently available from the IRIS Database. To derive an oral reference dose (RfD) for PFHxS, available toxicity studies were reviewed using a weight-of-evidence approach. A 42-day mouse reproductive study was chosen as the critical study for the derivation of the oral RfD. Benchmark dose modeling was utilized to derive a point of departure (POD) for a reduction in litter size. A 95% lower confidence limit on the benchmark dose (BMDL) of 13,900 ng/mL (serum PFHxS) was modeled for a reduction in litter size. An oral RfD for PFHxS of 4.0 ng/kg/d was calculated by conversion of the BMDL to a human equivalent oral dose using a human half-life adjusted dosimetric conversion factor and the application of a total uncertainty factor of 300. Additional research is needed to better characterize the toxicity associated with oral exposure to PFHxS and refine the development of toxicity values.

Bioaccessibility of polychlorinated dioxins and furans in soil from a Superfund site.

Bioaccessibilities of PCDD/PCDF congeners contributing to cancer risk were determined in twelve soil samples from the American Creosote Works Superfund site in Florida. Based upon sample locations, congener profiles (i.e., the same dominant congeners), and total (Toxic Equivalent; TEQ) concentrations, each of these samples has PCDD/PCDF contamination reasonably attributable to the site. Bioaccessibility was determined using a 2-phase in vitro extraction method that included both simulated gastric and intestinal conditions of the human GI tract. Measured congener-specific bioaccessibility values ranged from 34.3 to 62.1%. There was no apparent relationship between the extent of chlorination of PCDD/PCDF congeners and their bioaccessibility. TEQ-weighted bioaccessibility values varied among individual soil samples, which is not unexpected based upon the literature. This variability could not be explained by differences in soil pH, composition, or organic carbon content. The average TEQ-weighted bioaccessibility value of 59% for the twelve samples was accepted as representing site-specific bioavailability of PCDD/PCDFs. This value is higher than most dioxin/furan bioaccessibility values reported in the literature and at the upper end of the range of relative oral bioavailability (RBA) values reported for PCDD/PCDFs from in vivo bioavailability studies. This study used a finer fraction of soil particles (<150 microns versus the more typical <250 microns) to better represent soil that is incidentally ingested. This finer fraction would be expected to have a greater surface area available for extraction of PCDD/PCDFs per unit mass, which might account for the greater than expected bioaccessibility.

Influence of airborne particulates on respiratory tract deposition of inhaled toluene and naphthalene in the rat.

OBJECTIVE: Most studies report that inhaled volatile and semivolatile organic compounds (VOCs/SVOCs) tend to deposit in the upper respiratory tract, while ultrafine (or near ultrafine) particulate matter (PM) (∼100 nm) reaches the lower airways. The objective of this study was to determine whether carbon particle co-exposure carries VOCs/SVOCs deeper into the lungs where they are deposited. MATERIALS AND METHODS: Male Sprague-Dawley rats were exposed by inhalation (nose-only) to radiolabeled toluene (20 ppm) or naphthalene (20 ppm) on a single occasion for 1 h, with or without concurrent carbon particle exposure (∼5 mg/m3). The distribution of radiolabel deposited within the respiratory tract of each animal was determined after sacrifice. The extent of adsorption of toluene and naphthalene to airborne carbon particles under the exposure conditions of the study was also assessed. RESULTS: We found that in the absence of particles, the highest deposition of both naphthalene and toluene was observed in the upper respiratory tract. Co-exposure with carbon particles tended to increase naphthalene deposition slightly throughout the respiratory tract, whereas slight decreases in toluene deposition were observed. Few differences were statistically significant. Naphthalene showed greater adsorption to the particles compared to toluene, but overall the particle-adsorbed concentration of each of these compounds was a small fraction of the total inspired concentration. CONCLUSIONS: These studies imply that at the concentrations used for the exposures in this study, inhaled carbon particles do not substantially alter the deposition of naphthalene and toluene within the respiratory tract.

Targeted heat activation of HSP promoters in the skin of mammalian animals and humans.

The use of highly inducible HSP promoters for exerting spatial and/or temporal control over the expression of therapeutic transgenes has long been discussed. Localized and time-limited induction of the heat shock response may potentially also be of medical interest. However, such applications would require targeted delivery of heat doses capable of activating HSP promoters in tissues or organs of interest. Accessible areas, including the skin and tissues immediately underneath it, may be most readily targeted. A few applications for heat-directed or heat-controlled therapy in the skin might involve expression of proteins to restore or protect normal skin function, protein antigens for vaccination/immunotherapy, vaccine viruses or even systemically active proteins, e.g., cytokines and chemokines. A review of the literature relating to localized heat activation of HSP promoters and HSP genes in the skin revealed that a multitude of different technologies has been explored in small animal models. In contrast, we uncovered few publications that examine HSP promoter activation in human skin. None of these publications has a therapeutic focus. We present herein two, clinically relevant, developments of heating technologies that effectively activate HSP promoters in targeted regions of human skin. The first development advances a system that is capable of reliably activating HSP promoters in human scalp, in particular in hair follicles. The second development outlines a simple, robust, and inexpensive methodology for locally activating HSP promoters in small, defined skin areas.

Incremental Sampling Methodology: Applications for Background Screening Assessments.

This article presents the findings from a numerical simulation study that was conducted to evaluate the performance of alternative statistical analysis methods for background screening assessments when data sets are generated with incremental sampling methods (ISMs). A wide range of background and site conditions are represented in order to test different ISM sampling designs. Both hypothesis tests and upper tolerance limit (UTL) screening methods were implemented following U.S. Environmental Protection Agency (USEPA) guidance for specifying error rates. The simulations show that hypothesis testing using two-sample t-tests can meet standard performance criteria under a wide range of conditions, even with relatively small sample sizes. Key factors that affect the performance include unequal population variances and small absolute differences in population means. UTL methods are generally not recommended due to conceptual limitations in the technique when applied to ISM data sets from single decision units and due to insufficient power given standard statistical sample sizes from ISM.

Molecular Mechanisms of Perfluorooctanoate-Induced Hepatocyte Apoptosis in Mice Using Proteomic Techniques.

The stability of perfluorooctanoate (PFOA) coupled with its wide use cause serious concerns regarding its potential risk to human health. The molecular mechanisms of PFOA-induced hepatotoxicity relevant to human health was investigated using both in vivo (mouse model) and in vitro (human hepatocyte cells, HL-7702) techniques. Both male and female Balb/c mice were administered PFOA at 0.05, 0.5, or 2.5 mg/kg-d for 28-d, with serum PFOA concentrations after exposure being found at environmentally relevant levels. Liver samples were examined for histology and proteomic change using iTRAQ and Western Blotting, showing dose-dependent hepatocyte apoptosis and peroxisome proliferation. At high doses, genotoxicity resulting from ROS hypergeneration was due to suppression of Complex I subunits in the electron transport chain and activation of PPARα in both genders. However, at 0.05 mg/kg-d, Complex I suppression occurred only in females, making them more sensitive to PFOA-induced apoptosis. In vitro assays using HL-7702 cells confirmed that apoptosis was also induced through a similar mechanism. The dose/gender-dependent toxicity mechanisms help to explain some epidemiological phenomena, i.e., liver cancer is not often associated with PFOA exposure in professional workers. Our results demonstrated that a proteomic approach is a robust tool to explore molecular mechanisms of toxic chemicals at environmentally relevant levels.

Effects of Source and Concentration on Relative Oral Bioavailability of Benzo(a)pyrene from Soil.

The objective of this study was to examine the influence of soil composition, PAH concentration, and source material type on PAH bioavailability using an approach capable of measuring uptake at low, environmentally relevant PAH concentrations (down to 1 ppm). Contaminated soil samples were constructed using PAHs from three source materials-solvent, soot, and fuel oil-to which 3H-benzo(a)pyrene (3H-BaP; total BaP concentrations of 1, 10, and 100 ppm) was added in a mixture of PAHs. The soils were weathered for 8 weeks using weekly wet-dry cycles. Each soil was administered as a single dose to rats, and blood samples were taken over 6 days. Relative oral bioavailability (RBA) of the BaP from soil was estimated by comparing the area under the curve (AUC) for 3H concentration versus time in blood with the AUC observed from the same PAH mixture dosed in a food matrix. The extent to which BaP RBA was diminished in soil versus food varied among the source materials, but little or no difference was observed among the soil types examined unless carbon amendments were added. These results suggest that the type of PAH source material can have a strong influence on PAH oral bioavailability.

Applied Nanotoxicology.

Nanomaterials, including nanoparticles and nanoobjects, are being incorporated into everyday products at an increasing rate. These products include consumer products of interest to toxicologists such as pharmaceuticals, cosmetics, food, food packaging, household products, and so on. The manufacturing of products containing or utilizing nanomaterials in their composition may also present potential toxicologic concerns in the workplace. The molecular complexity and composition of these nanomaterials are ever increasing, and the means and methods being applied to characterize and perform useful toxicologic assessments are rapidly advancing. This article includes presentations by experienced toxicologists in the nanotoxicology community who are focused on the applied aspect of the discipline toward supporting state of the art toxicologic assessments for food products and packaging, pharmaceuticals and medical devices, inhaled nanoparticle and gastrointestinal exposures, and addressing occupational safety and health issues and concerns. This symposium overview article summarizes 5 talks that were presented at the 35th Annual meeting of the American College of Toxicology on the subject of "Applied Nanotechnology."

Oral Bioavailability, Bioaccessibility, and Dermal Absorption of PAHs from Soil-State of the Science.

This article reviews the state of the science regarding oral bioavailability, bioaccessibility, and dermal absorption of carcinogenic polycyclic aromatic hydrocarbons (cPAHs) in soil by humans, and discusses how chemical interactions may control the extent of absorption. Derived from natural and anthropomorphic origins, PAHs occur in a limited number of solid and fluid matrices (i.e., PAH sources) with defined physical characteristics and PAH compositions. Existing studies provide a strong basis for establishing that oral bioavailability of cPAHs from soil is less than from diet, and an assumption of 100% relative bioavailability likely overestimates exposure to cPAHs upon ingestion of PAH-contaminated soil. For both the oral bioavailability and dermal absorption studies, the aggregate data do not provide a broad understanding of how different PAH source materials, PAH concentrations, or soil chemistries influence the absorption of cPAHs from soil. This article summarizes the existing studies, identifies data gaps, and provides recommendations for the direction of future research to support new default or site-specific bioavailability adjustments for use in human health risk assessment.

Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth.

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.

Oral absorption of PEG-coated versus uncoated gold nanospheres: does agglomeration matter?

BACKGROUND: Particle size is thought to be a critical factor affecting the bioavailability of nanoparticles following oral exposure. Nearly all studies of nanoparticle bioavailability focus on characterization of the primary particle size of the material as supplied or as dosed, and not on agglomeration behavior within the gastrointestinal tract, which is presumably most relevant for absorption. METHODS: In the study reported here, snapshots of agglomeration behavior of gold nanospheres were evaluated in vivo throughout the gastrointestinal tract using transmission electron microscopy. Agglomeration state within the gastrointestinal tract was then used to help explain differences in gastrointestinal particle absorption, as indicated by tissue levels of gold detected using inductively coupled plasma mass spectrometry. Mice were dosed (10 mg/kg) with either 23 nm PEG-coated or uncoated gold nanospheres. RESULTS: Transmission electron microscopy demonstrates that PEG-coated gold nanoparticles can be observed as primary, un-agglomerated particles throughout the gastrointestinal tract and feces of dosed animals. In contrast, uncoated gold nanoparticles were observed to form agglomerates of several hundred nanometers in all tissues and feces. Inductively coupled plasma mass spectrometry shows significantly higher levels of gold in tissues from animals dosed with PEG-coated versus uncoated 23 nm gold nanoparticles. Retention of particles after a single oral gavage was also very high, with all tissues of animals dosed with PEG-coated particles having detectable levels of gold at 30 days following exposure. CONCLUSIONS: Qualitative observation of these particles in vivo shows that dispersed PEG-coated particles are able to reach the absorptive tissues of the intestine while agglomerated uncoated particles are sequestered in the lumen of these tissues. However, the large differences observed for in vivo agglomeration behavior were not reflected in oral absorption, as indicated by gold tissue levels. Additional factors, such as surface chemistry, may have played a more important role than in vivo particle size and should be investigated further.

Differential interferences with clinical chemistry assays by gold nanorods, and gold and silica nanospheres.

Nanomaterials are known to cause interference with several standard toxicological assays. As part of an in vivo study of PEG-coated gold nanorods in mice, nanorods were added to reference serum, and results for standard clinical chemistry parameters were compared with serum analyzed without nanorods. PEG-coated gold nanorods produced several concentration-dependent interferences. Comparisons were then made with PEG-coated gold and silica nanospheres. Interferences were observed for both materials that differed from gold nanorods. Removal of the particles from serum by centrifugation prior to analysis resolved most, but not all of the interferences. Additional clinical chemistry analyzers were used to further investigate trends in assay interference. We conclude that PEG-coated gold and silica nanoparticles can interfere with standard clinical chemistry tests in ways that vary depending upon material, shape, and specific assay methodology employed. Assay interferences by nanomaterials cannot always be predicted, underscoring the need to verify that nanomaterials under study do not interfere with methods used to evaluate potential biological effects.