An expert-driven literature review of "negative" chemicals for developmental neurotoxicity (DNT) in vitro assay evaluation.

To date, approximately 200 chemicals have been tested in US Environmental Protection Agency (EPA) or Organization for Economic Co-operation and Development (OECD) developmental neurotoxicity (DNT) guideline studies, leaving thousands of chemicals without traditional animal information on DNT hazard potential. To address this data gap, a battery of in vitro DNT new approach methodologies (NAMs) has been proposed. Evaluation of the performance of this battery will increase the confidence in its use to determine DNT chemical hazards. One approach to evaluate DNT NAM performance is to use a set of chemicals to evaluate sensitivity and specificity. Since a list of chemicals with potential evidence of in vivo DNT has been established, this study aims to develop a curated list of "negative" chemicals for inclusion in a "DNT NAM evaluation set". A workflow, including a literature search followed by an expert-driven literature review, was used to systematically screen 39 chemicals for lack of DNT effect. Expert panel members evaluated the scientific robustness of relevant studies to inform chemical categorizations. Following review, the panel discussed each chemical and made categorical determinations of "Favorable", "Not Favorable", or "Indeterminate" reflecting acceptance, lack of suitability, or uncertainty given specific limitations and considerations, respectively. The panel determined that 10, 22, and 7 chemicals met the criteria for "Favorable", "Not Favorable", and "Indeterminate", for use as negatives in a DNT NAM evaluation set. Ultimately, this approach not only supports DNT NAM performance evaluation but also highlights challenges in identifying large numbers of negative DNT chemicals.

An EPA database on the effects of engineered nanomaterials-NaKnowBase.

The US EPA Office of Research and Development (ORD) has conducted a research program assessing potential risks of emerging materials and technologies, including engineered nanomaterials (ENM). As a component of that program, a nanomaterial knowledge base, termed "NaKnowBase", was developed containing the results of published ORD research relevant to the potential environmental and biological actions of ENM. The experimental data address issues such as ENM release into the environment; fate, transport and transformations in environmental media; exposure to ecological species or humans; and the potential for effects on those species. The database captures information on the physicochemical properties of ENM tested, assays performed and their parameters, and the results obtained. NaKnowBase (NKB) is a relational SQL database, and may be queried either with SQL code or through a user-friendly web interface. Filtered results may be output in spreadsheet format for subsequent user-defined analyses. Potential uses of the data might include input to quantitative structure-activity relationships (QSAR), meta-analyses, or other investigative approaches.

Lack of Detectable Direct Effects of Silver and Silver Nanoparticles on Mitochondria in Mouse Hepatocytes.

Silver nanoparticles (AgNPs) are well-proven antimicrobial nanomaterials, yet little is elucidated regarding the mechanism underlying cytotoxicity induced by these nanoparticles. Here, we tested the hypothesis that mitochondria are primary intracellular targets of two AgNPs and silver ions in mouse hepatocytes (AML12) cultured in glucose- and galactose-based media. AML12 cells were more sensitive to mitochondrial uncoupling when grown with galactose rather than glucose. However, 24 h treatments with 15 nm AgNPs and 6 nm GA-AgNPs (5 and 10 µg/mL) and AgNO3 (1 and 3 µg/mL), concentrations that resulted in either 10 or 30% cytotoxicity, failed to cause more toxicity to AML12 cells grown on galactose than glucose. Furthermore, colocalization analysis and subcellular Ag quantification did not show any enrichment of silver content in mitochondria in either medium. Finally, the effects of the same exposures on mitochondrial respiration were mild or undetectable, a result inconsistent with mitochondrial toxicity causing cell death. Our results suggest that neither ionic Ag nor the AgNPs that we tested specifically target mitochondria and are inconsistent with mitochondrial dysfunction being the primary cause of cell death after Ag exposure under these conditions.

The effects of gene × environment interactions on silver nanoparticle toxicity in the respiratory system: An adverse outcome pathway.

The Adverse Outcome Pathway (AOP) framework is serving as a basis to integrate new data streams in order to enhance the power of predictive toxicology. AOP development for engineered nanomaterials (ENM), including silver nanoparticles (AgNP), is currently lagging behind other chemicals of regulatory interest due to our limited understanding of the mechanism by which underlying genetics or diseases directly modify host response to AgNP exposures. This also highlights the importance of considering the Aggregate Exposure Pathway (AEP) framework, which precedes the AOP framework and outlines source to target site exposure. The AEP and AOP frameworks interface at the target site, where a molecular initiating event (MIE) occurs and is followed by key events (KE) for adverse cellular and organ responses along a biological pathway and ends with the adverse organism response. The primary goal of this study is to use AgNP to interrogate the AEP-AOP framework by organizing and integrating in vitro dose-response data and in vivo exposure-response data from previous studies to evaluate the effects of interactions between host genetic and acquired factors, or gene × environment interactions (G × E), on AgNP toxicity in the respiratory system. Using this framework will help us to identify plausible key event relationships (KER) between MIE and adverse organism responses when KE are not measured using the same assay in order to derive future predictive models, guide research, and support development of tools for making risk-based, regulatory decisions on ENM. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

3D Printer Particle Emissions: Translation to Internal Dose in Adults and Children.

Desktop fused deposition modeling (FDM®) three-dimensional (3D) printers are becoming increasingly popular in schools, libraries, and among home hobbyists. FDM® 3D printers have been shown to release ultrafine airborne particles in large amounts, indicating the potential for inhalation exposure and consequent health risks among FDM® 3D printer users and other room occupants including children. These particles are generated from the heating of thermoplastic polymer feedstocks during the FDM® 3D printing process, with the most commonly used polymers being acrylonitrile butadiene styrene (ABS) and poly-lactic acid (PLA). Risk assessment of these exposures demands estimation of internal dose, especially to address intra-human variability across life stages. Dosimetry models have proven to effectively translate particle exposures to internal dose metrics relevant to evaluation of their effects in the respiratory tract. We used the open-access multiple path particle dosimetry (MPPD v3.04) model to estimate inhaled particle deposition in different regions of the respiratory tract for children of various age groups from three months to eighteen years old adults. Mass concentration data for input into the MPPD model were calculated using particle size distribution and density data from experimental FDM® 3D printer emissions tests using both ABS and PLA. The impact of changes in critical parameters that are principal determinants of inhaled dose, including: sex, age, and exposure duration, was examined using input parameter values available from the International Commission on Radiological Protection. Internal dose metrics used included regional mass deposition, mass deposition normalized by pulmonary surface area, surface area of deposited particles by pulmonary surface area, and retained regional mass. Total mass deposition was found to be highest in the 9-year-old to 18-year-old age groups with mass deposition by pulmonary surface area highest in 3-month-olds to 9-year-olds and surface area of deposited particles by pulmonary surface area to be highest in 9-year-olds. Clearance modeling revealed that frequent 3D printer users are at risk for an increased cumulative retained dose.

Detection of large extracellular silver nanoparticle rings observed during mitosis using darkfield microscopy.

During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 µg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after treatment. Approximately cell-sized extracellular rings of deposited AgNP were observed on the slides among a field of dispersed individual AgNP. The mean diameter of 45 nanoparticles circles was 62.5 +/-12 microns. Ring structures were frequently observed near what appeared to be post-mitotic daughter cells, giving rise to the possibility that cell membrane fragments were deposited on the slide during mitosis, and those fragments selectively attracted and retained silver nanoparticles from suspension in the cell culture medium. These circular structures were observable for the following technical reasons: 1) darkfield microscope could observe single nanoparticles below 100 nm in size, 2) a large concentration (108 and 109) of nanoparticles was used in these experiments 3) negatively charged nanoparticles were attracted to adhesion membrane proteins remaining on the slide from mitosis. The observation of silver nanoparticles attracted to apparent remnants of cellular mitosis could be a useful tool for the study of normal and abnormal mitosis.

Particle and volatile organic compound emissions from a 3D printer filament extruder.

Fused Deposition Modeling (FDM®), also known as Fused Filament Fabrication (FFF), 3D printers have been shown in numerous studies to emit ultrafine particles and volatile organic compounds (VOCs). Filament extruders, designed to create feedstocks for 3D printers, have recently come onto the consumer market for at-home hobbyists as an alternative to buying 3D printer filaments. These instruments allow for the creation of 3D printer filaments from raw plastic pellets. Given the similarity in processes and materials used by 3D printers and filament extruders, we hypothesized that filament extruders may also release ultrafine particle emissions and VOCs. An off-the-shelf filament extruder was operated in a 2 m3 chamber using three separate feedstocks: acrylonitrile butadiene styrene (ABS) pellets, pulverized poly-lactic acid (PLA), and PLA pellets. Ultrafine particle emissions were measured in real-time using a scanning mobility particle sizer and thermal desorption tubes were used for both non-targeted and targeted analysis of VOCs present in emissions. Ultrafine particle number emission rates were comparable to those found in 3D printer studies with the greatest to least emission rates from ABS pellets, pulverized PLA, and PLA pellets, respectively. In addition, the majority of particles released were found to be ultrafine (1-100 nm), similar to 3D printer studies. A variety of VOCs were identified using the ABS feedstock, including styrene and ethylbenzene, and PLA feedstock. Styrene average mass concentration amounts were found to be near the EPA Integrated Risk Information System Reference Concentration for Inhalation Exposure for 3 min and 5 min samples. Further studies will be needed to determine the impact on emissions of environmental volume, air exchange rate, and extruder settings such as extrusion speed and temperature. The results support the hypothesis that use of a filament extruder may present an additional exposure risk to 3D printer hobbyists.

Neurotoxicology of Nanomaterials.

The remarkable advances coming about through nanotechnology promise to revolutionize many aspects of modern life; however, these advances come with a responsibility for due diligence to ensure that they are not accompanied by adverse consequences for human health or the environment. Many novel nanomaterials (having at least one dimension <100 nm) could be highly mobile if released into the environment and are also very reactive, which has raised concerns for potential adverse impacts including, among others, the potential for neurotoxicity. Several lines of evidence led to concerns for neurotoxicity, but perhaps none more than observations that inhaled nanoparticles impinging on the mucosal surface of the nasal epithelium could be internalized into olfactory receptor neurons and transported by axoplasmic transport into the olfactory bulbs without crossing the blood-brain barrier. From the olfactory bulb, there is concern that nanomaterials may be transported deeper into the brain and affect other brain structures. Of course, people will not be exposed to only engineered nanomaterials, but rather such exposures will occur in a complex mixture of environmental materials, some of which are incidentally generated particles of a similar inhalable size range to engineered nanomaterials. To date, most experimental studies of potential neurotoxicity of nanomaterials have not considered the potential exposure sources and pathways that could lead to exposure, and most studies of nanomaterial exposure have not considered potential neurotoxicity. Here, we present a review of potential sources of exposures to nanoparticles, along with a review of the literature on potential neurotoxicity of nanomaterials. We employ the linked concepts of an aggregate exposure pathway (AEP) and an adverse outcome pathway (AOP) to organize and present the material. The AEP includes a sequence of key events progressing from material sources, release to environmental media, external exposure, internal exposure, and distribution to the target site. The AOP begins with toxicant at the target site causing a molecular initiating event and, like the AEP, progress sequentially to actions at the level of the cell, organ, individual, and population. Reports of nanomaterial actions are described at every key event along the AEP and AOP, except for changes in exposed populations that have not yet been observed. At this last stage, however, there is ample evidence of population level effects from exposure to ambient air particles that may act similarly to engineered nanomaterials. The data give an overall impression that current exposure levels may be considerably lower than those reported experimentally to be neurotoxic. This impression, however, is tempered by the absence of long-term exposure studies with realistic routes and levels of exposure to address concerns for chronic accumulation of materials or damage. Further, missing across the board are "key event relationships", which are quantitative expressions linking the key events of either the AEP or the AOP, making it impossible to quantitatively project the likelihood of adverse neurotoxic effects from exposure to nanomaterials or to estimate margins of exposure for such relationships.

Combination of Dark-Field and Confocal Microscopy for the Optical Detection of Silver and Titanium Nanoparticles in Mammalian Cells.

We describe here two optical microscopy techniques-dark-field confocal light scanning microscopy (DF-CLSM) and dark-field wide-field confocal microscopy (DF-WFCM), that can be used to study interaction between nanoparticles and cells in 3D space. Dark field microscopy can detect very small structures below the diffraction limit of conventional light microscopes, while a confocal setup provides vertical sectioning capabilities to render specimens in 3D. The use of DF-WFCM instead of DF-CLSM allows faster sample processing but yields lower resolution. We used a retinal pigment epithelial cell line ARPE-19 to illustrate different optical and lighting conditions necessary for optimal imaging of metal and metal oxide nanoparticles (TiO2 and Ag). Our experimental setup primarily involved an E-800 Nikon and Nikon Ni upright microscopes and a Nikon Ti2 microscope connected to a xenon light source along with special dark-field objectives. For confocal studies we used either Leica and Nikon inverted confocal microscopes. For microscopic analyses, ARPE-19 cells were fixed in situ in cultured chamber slides or collected from T-25 flasks and then fixed in suspension. At the lowest concentrations of TiO2 or Ag tested (0.1-0.3 µg/mL), we were able to detect as few as 5-10 nanoparticles per cell due to intense light scattering by the particles. The degree of brightness detected indicated that the uptake of nanoparticles within ARPE-19 cells could be monitored using dark-field microscopy. Here we describe how to use wide-field microscopy to follow nanoparticle uptake by cells and how to assess some aspects of cellular health in in vitro cell cultures exposed to nanoparticles.

Detection of Silver and TiO2 Nanoparticles in Cells by Flow Cytometry.

Evaluation of the potential hazard of man-made nanomaterials has been hampered by a limited ability to observe and measure nanoparticles in cells. A FACSCalibur™ flow cytometer and a Stratedigm S-1000 flow cytometer were used to measure changes in light scatter from cells after incubation with either silver nanoparticles (AgNP) or TiO2 nanoparticles. Within the range of between 0.1 µg/mL and 30 µg/mL the nanoparticles caused a proportional increase of the side scatter and decrease of the forward scatter intensity signals. At the lowest concentrations of TiO2 (ranging between 0.1 µg/mL and 0.3 µg/mL), the flow cytometer can detect as few as 5-10 nanoparticles per cell. The influence of nanoparticles on the cell cycle was detected by nonionic detergent lysis of nanoparticle incubated cells that were stained with DAPI or propidium iodide (PI). Viability of nanoparticle treated cells was determined by PI exclusion. Surface plasmonic resonance (SPR) was detected primarily in the far-red fluorescence detection channels after excitation with a 488 nm laser.Our results suggest that the uptake of nanoparticles within cells can be monitored using flow cytometry. This uptake of nanoparticle data was confirmed by viewing the nanoparticles in the cells using dark-field microscopy. The flow cytometry detection of nanoparticles approach may help fill a critical need to assess the relationship between nanoparticle dose and cellular toxicity. Such experiments using nanoparticles could potentially be performed quickly and easily using the flow cytometer to measure both nanoparticle uptake and cellular health.

Biophysical comparison of four silver nanoparticles coatings using microscopy, hyperspectral imaging and flow cytometry.

This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G1 phase and an increase in the G2/M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions.

Particle emissions from fused deposition modeling 3D printers: Evaluation and meta-analysis.

Fused deposition modeling (FDM) 3D printers, the most popular choice among home hobbyists, have been shown to release volatile organic chemicals (VOCs) and billions of airborne particles per minute, indicating the potential for consumer inhalation exposure and consequent health risks. Publications on FDM 3D printer emissions however, contain large heterogeneity of testing methods and analytical procedures making it difficult to reach overall conclusions for particle characteristics or particle number emission rates across the field. In this publication, data were collected over the printing time from 3D printer emission studies including particle count diameters (PCDs) (nanometers), particle number concentrations (PNCs) (particles/cm3), and particle number emission rates (PNERs) (particles min-1). Despite heterogeneity in methods, the majority of particles released were reported as ultrafine in size (i.e., <100 nm) indicating that using both acrylonitrile butadiene styrene (ABS) and poly-lactic acid (PLA) may present a risk of exposure to respirable particles. Mean PNC emitted in 3D printing tests ranged over several orders of magnitude across publications with overall means of 300,980 particles/cm3 for ABS and 65,482 particles/cm3 for PLA. Although mean PNC data were available from only 7 of the 16 papers reviewed, ABS resulted in greater particle numbers than PLA suggesting increased exposure to ultrafine particles. A linear mixed model was fitted for mean PNCs to further explore the impact of nozzle temperature and filament material. Finally, the PNER calculation method especially regarding losses, varied widely across studies, and directly impacted the PNERs reported. To strengthen direct comparability of results going forward, it is recommended that standard emissions testing protocols be developed for FDM 3D printers and particle influxes and losses be more uniformly calculated.

Determination of Silver Nanoparticle Dose in vitro.

An important issue for interpreting in vitro nanomaterial testing is quantifying the dose delivered to target cells. Considerations include the concentration added to the culture, the proportion of the applied dose that interacts with the target cells, and the amount that is eventually absorbed by the target cells. Rapid and efficient techniques are needed to determine delivered doses. Previously, we demonstrated that TiO2 and silver nanoparticles (AgNP) were absorbed by cells in a dose dependent manner between 1 µg/ml and 30 µg/ml and were detected in cells by light scatter using a flow cytometer. Here, we compare four potential indices of the dose of AgNP to cells, including: inductively coupled plasma - mass spectrometry (ICP-MS); flow cytometry side scatter (SSC); and amount of silver deposited to the cell layer as estimated with both an integrated Volumetric Centrifugation Method - In Vitro Sedimentation, Diffusion and Dosimetry Model (VCM-ISDD) and a Distorted Grid (DG) model. A retinal pigment epithelial cell line was exposed to 20 nm or 75 nm citrate-coated AgNP for 24 hr. The relationships between particle sizes and internalized doses varied according to the dose metric. Twenty-four hours after exposure, the cell layer contained a greater mass of silver when treated with 75 nm AgNP than with 20 nm AgNP. When the dose was expressed as the number of particles or as the total surface area of absorbed particles, however, the reverse was true; the dose to the cells was higher after exposure to 20 than 75 nm AgNP. Flow cytometry SSC increased with dose for both sizes of AgNP, and was correlated with Ag in cells measured by ICP-MS. The rate of SSC increase was greater for 75 than for 20 nm AgNP, suggesting it could be used as an indicator of cellular dose after accounting for particle size and composition. Silver was detected by ICP-MS in re-suspended supernates of the isolated cell layer suggested that not all the silver deposited to the cell layer was absorbed by the cells. Both the VCM-ISDD and DG models estimated the proportion of Ag deposited to the cellular layer, which in both cases was greater than the amount of silver in the cells measured by ICP-MS. Modeled deposition more closely compared to the total Ag deposition by ICP-MS, i.e. mass of silver in the cells plus the resuspended, unabsorbed Ag from the cell layer. ICP-MS indicated the mass of silver in cells from AgNP treatment, but not whether the Ag was in the form of particles or dissolved ions. Deposition models predicted the amount of AgNP deposited to the cell layer, but not cellular uptake. Flow cytometry SSC was correlated to cellular uptake of particle-form AgNP and could be calibrated against ICP-MS to indicate mass of cellular uptake. Therefore, a combination of approaches may be required to accurately understand cellular dosimetry of in vitro nanotoxicology experiments. In summary, cellular dosimetry is an important consideration for nanotoxicology experiments, and not necessarily related to the applied dose.

Moderate perinatal thyroid hormone insufficiency alters visual system function in adult rats.

Thyroid hormone (TH) is critical for many aspects of neurodevelopment and can be disrupted by a variety of environmental contaminants. Sensory systems, including audition and vision are vulnerable to TH insufficiencies, but little data are available on visual system development at less than severe levels of TH deprivation. The goal of the current experiments was to explore dose-response relations between graded levels of TH insufficiency during development and the visual function of adult offspring. Pregnant Long Evans rats received 0 or 3 ppm (Experiment 1), or 0, 1, 2, or 3 ppm (Experiment 2) of propylthiouracil (PTU), an inhibitor of thyroid hormone synthesis, in drinking water from gestation day (GD) 6 to postnatal day (PN) 21. Treatment with PTU caused dose-related reductions of serum T4, with recovery on termination of exposure, and euthyroidism by the time of visual function testing. Tests of retinal (electroretinograms; ERGs) and visual cortex (visual evoked potentials; VEPs) function were assessed in adult offspring. Dark-adapted ERG a-waves, reflecting rod photoreceptors, were increased in amplitude by PTU. Light-adapted green flicker ERGs, reflecting M-cone photoreceptors, were reduced by PTU exposure. UV-flicker ERGs, reflecting S-cones, were not altered. Pattern-elicited VEPs were significantly reduced by 2 and 3 ppm PTU across a range of stimulus contrast values. The slope of VEP amplitude-log contrast functions was reduced by PTU, suggesting impaired visual contrast gain. Visual contrast gain primarily reflects function of visual cortex, and is responsible for adjusting sensitivity of perceptual mechanisms in response to changing visual scenes. The results indicate that moderate levels of pre-and post-natal TH insufficiency led to alterations in visual function of adult rats, including both retinal and visual cortex sites of dysfunction.

Manganese testing under a clean air act test rule and the application of resultant data in risk assessments.

In the 1990's, the proposed use of methylcyclopentadienyl manganese tricarbonyl (MMT) as an octane-enhancing gasoline fuel additive led to concerns for potential public health consequences from exposure to manganese (Mn) combustion products in automotive exhaust. After a series of regulatory/legal actions and negotiations, the U.S. Environmental Protection Agency (EPA) issued under Clean Air Act (CAA) section 211(b) an Alternative Tier 2 Test Rule that required development of scientific information intended to help resolve uncertainties in exposure or health risk estimates associated with MMT use. Among the uncertainties identified were: the chemical forms of Mn emitted in automotive exhaust; the relative toxicity of different Mn species; the potential for exposure among sensitive subpopulations including females, the young and elderly; differences in sensitivity between test species and humans; differences between inhalation and oral exposures; and the influence of dose rate and exposure duration on tissue accumulation of Mn. It was anticipated that development of specific sets of pharmacokinetic (PK) information and models regarding Mn could help resolve many of the identified uncertainties and serve as the best foundation for available data integration. The results of the test program included development of several unique Mn datasets, and a series of increasingly sophisticated Mn physiologically-based pharmacokinetic (PBPK) models. These data and models have helped address each of the uncertainties originally identified in the Test Rule. The output from these PBPK models were used by the Agency for Toxic Substances and Disease Registry (ATSDR) in 2012 to inform the selection of uncertainty factors for deriving the manganese Minimum Risk Level (MRL) for chronic exposure durations. The EPA used the MRL in the Agency's 2015 evaluation of potential residual risks of airborne manganese released from ferroalloys production plants. This resultant set of scientific data and models likely would not exist without the CAA section 211(b) test rule regulatory procedure.

A comprehensive framework for evaluating the environmental health and safety implications of engineered nanomaterials.

Engineered nanomaterials (ENM) are a growing aspect of the global economy, and their safe and sustainable development, use, and eventual disposal requires the capability to forecast and avoid potential problems. This review provides a framework to evaluate the health and safety implications of ENM releases into the environment, including purposeful releases such as for antimicrobial sprays or nano-enabled pesticides, and inadvertent releases as a consequence of other intended applications. Considerations encompass product life cycles, environmental media, exposed populations, and possible adverse outcomes. This framework is presented as a series of compartmental flow diagrams that serve as a basis to help derive future quantitative predictive models, guide research, and support development of tools for making risk-based decisions. After use, ENM are not expected to remain in their original form due to reactivity and/or propensity for hetero-agglomeration in environmental media. Therefore, emphasis is placed on characterizing ENM as they occur in environmental or biological matrices. In addition, predicting the activity of ENM in the environment is difficult due to the multiple dynamic interactions between the physical/chemical aspects of ENM and similarly complex environmental conditions. Others have proposed the use of simple predictive functional assays as an intermediate step to address the challenge of using physical/chemical properties to predict environmental fate and behavior of ENM. The nodes and interactions of the framework presented here reflect phase transitions that could be targets for development of such assays to estimate kinetic reaction rates and simplify model predictions. Application, refinement, and demonstration of this framework, along with an associated knowledgebase that includes targeted functional assay data, will allow better de novo predictions of potential exposures and adverse outcomes.

Toluene inhalation exposure for 13 weeks causes persistent changes in electroretinograms of Long-Evans rats.

Studies of humans chronically exposed to volatile organic solvents have reported impaired visual functions, including low contrast sensitivity and reduced color discrimination. These reports, however, lacked confirmation from controlled laboratory experiments. To address this question experimentally, we examined visual function by recording visual evoked potentials (VEP) and/or electroretinograms (ERG) from four sets of rats exposed repeatedly to toluene. In addition, eyes of the rats were examined with an ophthalmoscope and some of the retinal tissues were evaluated for rod and M-cone photoreceptor immunohistochemistry. The first study examined rats following exposure to 0, 10, 100 or 1000ppm toluene by inhalation (6hr/d, 5d/wk) for 13 weeks. One week after the termination of exposure, the rats were implanted with chronically indwelling electrodes and the following week pattern-elicited VEPs were recorded. VEP amplitudes were not significantly changed by toluene exposure. Four to five weeks after completion of exposure, rats were dark-adapted overnight, anesthetized, and several sets of electroretinograms (ERG) were recorded. In dark-adapted ERGs recorded over a 5-log (cd-s/m(2)) range of flash luminance, b-wave amplitudes were significantly reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A second set of rats, exposed concurrently with the first set, was tested approximately one year after the termination of 13 weeks of exposure to toluene. Again, dark-adapted ERG b-wave amplitudes were reduced at high stimulus luminance values in rats previously exposed to 1000ppm toluene. A third set of rats was exposed to the same concentrations of toluene for only 4 weeks, and a fourth set of rats exposed to 0 or 1000ppm toluene for 4 weeks were tested approximately 1year after the completion of exposure. No statistically significant reductions of ERG b-wave amplitude were observed in either set of rats exposed for 4 weeks. No significant changes were observed in ERG a-wave amplitude or latency, b-wave latency, UV- or green-flicker ERGs, or in photopic flash ERGs. There were no changes in the density of rod or M-cone photoreceptors. The ERG b-wave reflects the firing patterns of on-bipolar cells. The reductions of b-wave amplitude after 13 weeks of exposure and persisting for 1year suggest that alterations may have occurred in the inner nuclear layer of the retina, where the bipolar cells reside, or the outer or inner plexiform layers where the bipolar cells make synaptic connections. These data provide experimental evidence that repeated exposure to toluene may lead to subtle persistent changes in visual function. The fact that toluene affected ERGs, but not VEPs, suggests that elements in the rat retina may be more sensitive to organic solvent exposure than the rat visual cortex.

Neurophysiological assessment of auditory, peripheral nerve, somatosensory, and visual system function after developmental exposure to gasoline, E15, and E85 vapors.

The use of gasolines blended with a range of ethanol concentrations may result in inhalation of vapors containing a variable combination of ethanol with other volatile gasoline constituents. The possibility of exposure and potential interactions between vapor constituents suggests the need to evaluate the possible risks of this complex mixture. Previously we evaluated the effects of developmental exposure to ethanol vapors on neurophysiological measures of sensory function as a component of a larger project evaluating developmental ethanol toxicity. Here we report an evaluation using the same battery of sensory function testing in offspring of pregnant dams exposed during gestation to condensed vapors of gasoline (E0), gasoline blended with 15% ethanol (E15) or gasoline blended with 85% ethanol (E85). Pregnant Long-Evans rats were exposed to target concentrations 0, 3000, 6000, or 9000 ppm total hydrocarbon vapors for 6.5h/day over GD9 - GD20. Sensory evaluations of male offspring began as adults. The electrophysiological testing battery included tests of: peripheral nerve (compound action potentials, nerve conduction velocity [NCV]), somatosensory (cortical and cerebellar evoked potentials), auditory (brainstem auditory evoked responses), and visual functions. Visual function assessment included pattern elicited visual evoked potentials (VEP), VEP contrast sensitivity, dark-adapted (scotopic) electroretinograms (ERGs), light-adapted (photopic) ERGs, and green flicker ERGs. The results included sporadic statistically significant effects, but the observations were not consistently concentration-related and appeared to be statistical Type 1 errors related to multiple dependent measures evaluated. The exposure concentrations were much higher than can be reasonably expected from typical exposures to the general population during refueling or other common exposure situations. Overall the results indicate that gestational exposure of male rats to ethanol/gasoline vapor combinations did not cause detectable changes in peripheral nerve, somatosensory, auditory, or visual function when the offspring were assessed as adults.

Characterization, detection, and counting of metal nanoparticles using flow cytometry.

There is a need to accurately detect, characterize, and quantify nanoparticles in suspensions. This study helps to understand the complex interactions between similar types of nanoparticles. Before initiating a study of metal nanoparticles, five submicron PS beads with sizes between 200 nm and 1 µm were used to derive a reference scale that was useful in evaluating the flow cytometer for functionality, sensitivity, resolution, and reproducibility. Side scatter intensity (SSC) from metal nanoparticles was obtained simultaneously from 405 nm and 488 nm lasers. The 405 nm laser generally yielded histogram distributions with smaller CVs, less side scatter intensity, better separation indices between beads and decreased scatter differences between different sized particles compared with the 488 nm laser. Submicron particles must be diluted to 10(6) and 10(7) particles/mL before flow cytometer analysis to avoid coincidence counting artifacts. When particles were too concentrated the following occurred: swarm, electronic overload, coincidence counting, activation of doublet discrimination and rejection circuitry, increase of mean SSC histogram distributions, alterations of SSC and pulse width histogram shape, decrease and fluctuations in counting rate and decrease or elimination of particulate water noise and 1 µm reference bead. To insure that the concentrations were in the proper counting range, the nanoparticle samples were mixed with a known concentration of 1 µm counting beads. Sequential dilutions of metal nanoparticles in a 1 µm counting bead suspension helped determine the diluted concentration needed for flow cytometer analysis. It was found that the original concentrated nanoparticle samples had to be diluted, between 1:10,000 and 1:100,000, before characterization by flow cytometry. The concentration of silver or gold nanoparticles in the undiluted sample were determined by comparing them with a known concentration (1.9 × 10(6) beads/mL) of 1 µm polystyrene reference beads.

Use of novel inhalation kinetic studies to refine physiologically-based pharmacokinetic models for ethanol in non-pregnant and pregnant rats.

Ethanol (EtOH) exposure induces a variety of concentration-dependent neurological and developmental effects in the rat. Physiologically-based pharmacokinetic (PBPK) models have been used to predict the inhalation exposure concentrations necessary to produce blood EtOH concentrations (BEC) in the range associated with these effects. Previous laboratory reports often lacked sufficient detail to adequately simulate reported exposure scenarios associated with BECs in this range, or lacked data on the time-course of EtOH in target tissues (e.g. brain, liver, eye, fetus). To address these data gaps, inhalation studies were performed at 5000, 10 000, and 21 000 ppm (6 h/d) in non-pregnant female Long-Evans (LE) rats and at 21 000 ppm (6.33 h/d) for 12 d of gestation in pregnant LE rats to evaluate our previously published PBPK models at toxicologically-relevant blood and tissue concentrations. Additionally, nose-only and whole-body plethysmography studies were conducted to refine model descriptions of respiration and uptake within the respiratory tract. The resulting time-course and plethysmography data from these in vivo studies were compared to simulations from our previously published models, after which the models were recalibrated to improve descriptions of tissue dosimetry by accounting for dose-dependencies in pharmacokinetic behavior. Simulations using the recalibrated models reproduced these data from non-pregnant, pregnant, and fetal rats to within a factor of 2 or better across datasets, resulting in a suite of model structures suitable for simulation of a broad range of EtOH exposure scenarios.