Abstract  The report was a result of an effort by experts from government, industry and the public interest community to examine the path of a number of hypothetical nanotechnology food packaging applications through the current regulatory system. The regulatory system for food packaging is scientifically rigorous and extraordinarily complex, both legally and scientifically. This first-of-its-kind analysis provides a better understanding of the potential regulatory issues on the horizon for nanotechnology-enabled packaging – an advantage for industry, consumers and regulatory agencies such as FDA and the Environmental Protection Agency (EPA).

Abstract  There is an emerging literature reporting toxic effects of manufactured nanomaterials (NMs) and nanoparticles (NPs) in fish, but the mechanistic basis of both exposure and effect are poorly understood. This paper critically evaluates some of the founding assumptions in fish toxicology, and likely mechanisms of absorption, distribution, metabolism and excretion (ADME) of NPs in fish compared to other chemicals. Then, using a case study approach, the paper compares these assumptions for two different NPs; TiO2 and C60 fullerenes. Adsorption of NPs onto the gill surface will involve similar processes in the gill microenvironment and mucus layer to other substances, but the uptake mechanisms for NPs by epithelial cells are more likely to occur via vesicular processes (e.g., endocytosis) than uptake on membrane transporters or by diffusion through the cell membranes. Target organs may include the gills, gut, liver and sometimes the brain. Information on metabolism and excretion of NPs in fish is limited; but hepatic excretion into the bile seems a more likely mechanism, rather than mainly by renal or branchial excretion. TiO2 and C60 share some common chemical properties that appear to be associated with some similar toxic effects, but there are also differences, that highlight the notion that chemical reactivity can inform toxic effect of NPs in a fundamentally similar way to other chemicals. In this paper we identify many knowledge gaps including the lack of field observations on fish and other wildlife species for exposure and effects of manufactured NMs. Systematic studies of the abiotic factors that influence bioavailability, and investigation of the cell biology that informs on the mechanisms of metabolism and excretion of NMs, will greatly advance our understanding of the potential for adverse effects. There are also opportunities to apply existing tools and techniques to fundamental studies of fish toxicology with NPs, such as perfused organs and fish cell culture systems.

Abstract  It is inevitable that, during their use, engineered nanoparticles will be released into soils and waters. There is therefore increasing concern over the potential impacts of engineered nanoparticles in the environment on aquatic and terrestrial organisms and on human health. Once released into the environment, engineered nanoparticles will aggregate to some degree; they might also associate with suspended solids, sediment, be accumulated by organisms and enter drinking water sources and food materials. These fate processes are dependent on the characteristics of the particle and the characteristics of the environmental system. A range of ecotoxicological effects have also been reported, including effects on microbes, plants, invertebrates and fish. Although available data indicate that current risks of engineered nanoparticles in the environment to environmental and human health are probably low, our knowledge of the potential impacts of engineered nanoparticles in the environment on human health is still limited. There is therefore a need for continued work to develop an understanding of the exposure levels for engineered nanoparticles in environmental systems and to begin to explore the implications of these levels in terms of the ecosystem and human health. This will require research in a range of areas, including detection and characterization, environmental fate and transport, ecotoxicology and toxicology.

Abstract  This paper describes the issues relating to the measurement of nanoparticle size, shape and dispersion when evaluating the toxicity of nanoparticles. Complete characterization of these materials includes much more than size, size distribution and shape; nonetheless, these attributes are usually the essential foundation. The measurement of particle size, particularly at scales of 100 nm or less, can be challenging under the best of conditions. Measurements that are routine in the laboratory setting become even more difficult when made under the physiological conditions relevant to toxicity studies, where the environment of the particles can be quite complex. Passive and active cellular responses, as well as the presence of a variety of nano-scale biological structures, often complicate the collection and interpretation of size and shape data. In this paper, we highlight several of the common issues faced when characterizing nanoparticles for toxicity testing and suggest general protocols to address these problems.

Abstract  1. Isolated, non-identified neurons were voltage clamped using the internal perfusion technique. 2. Ions of Ag+ (1-100-mu-M) introduced into the bathing solution activated a steady-state inward current (I(Ag)) in the soma. The effect of Ag+ was reversible when the concentration of Ag+ was less than 75-mu-M or the time of alication was shorter than 10 min. 3. I(Ag) was observed both in the presence and absence of Na+ ions in the extracellular saline. It could also be activated when Cs+ ions were substituted for Na+ ions. 4. The current-voltage characteristics were linear in the voltage range - 100 to 0 mV. The revrsal potential in control saline was an average of 1.19 +/- 5.1 mV. 5. The application of Ag+ ions induces an elevation of intracellular free Ca2+ concentration by 10-20 times in both Ca2+-containing and Ca2+-free extracellular salines, as revealed by Fura-2 measurements. 6. Agents that increase the intracellular free Ca2+ concentration ([Ca2+]i), like thymol, caffeine and dinitrophenol, increased the amplitude of I(Ag). The effect was additive. Ruthenium Red, which blocks the release of Ca2+ from intracellular stores, decreased the Ag+ effect. 7. It is concluded that extracellularly applied Ag+ ions increase the cytoplasmic free Ca2+ concentration, which in turn activates non-specific cationic channels. 8. Ag+ ions in 1-10-mu-M concentration were able to decrease the voltage-activated Ca2+ current amplitude. This decrease, however, was due to the increase of [Ca2+]i which caused Ca2+-dependent inactivation.

Abstract  The cardiovascular system is currently considered a target for particulate matter, especially for ultrafine particles. In addition to autonomic or cytokine mediated effects, the direct interaction of inhaled materials with the target tissue must be examined to understand the underlying mechanisms. In the first approach, pulmonary and systemic distribution of inhaled ultrafine elemental silver (EAg) particles was investigated on the basis of morphology and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Rats were exposed for 6 hr at a concentration of 133 Ág EAg m3 (3 * 106 cm3, 15 nm modal diameter) and were sacrificed on days 0, 1, 4, and 7. ICP-MS analysis showed that 1.7 Ág Ag was found in the lungs immediately after the end of exposure. Amounts of Ag in the lungs decreased rapidly with time, and by day 7 only 4% of the initial burden remained. In the blood, significant amounts of Ag were detected on day 0 and thereafter decreased rapidly. In the liver, kidney, spleen, brain, and heart, low concentrations of Ag were observed. Nasal cavities, especially the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of Ag. For comparison, rats received by intratracheal instillation either 150 ÁL aqueous solution of 7 Ág silver nitrate (AgNO3) (4.4 Ág Ag) or 150 ÁL aqueous suspension of 50 Ág agglomerated ultrafine EAg particles. A portion of the agglomerates remained undissolved in the alveolar macrophages and in the septum for at least 7 days. In contrast, rapid clearance of instilled water-soluble AgNO3 from the lung was observed. These findings show that although instilled agglomerates of ultrafine EAg particles were retained in the lung, Ag was rapidly cleared from the lung after inhalation of ultrafine EAg particles, as well as after instillation of AgNO3, and entered systemic pathways.

Abstract  Algal bioassays for heavy metals can detect low levels in the environment, for example, 0.01 ppm for silver. Algae respond to increasing levels of heavy metals such as copper, nickel, mercury, silver, or cadmium by reduction of growth rate. Occasionally, the response to nontoxic metals is an increase in growth rate. At very low concentrations some potentially toxic metals may be necessary micronutrients. Algal species differ quite markedly in their sensitivity to heavy metals. Combined effects of two or more metals at toxic concentrations may be synergistic (for example, copper-nickel) or antagonistic (for example, cadmium-selenium). The critical concentrations for toxicity of a particular metal may be different at different times during the growth of an algal culture, as well as being dependent upon other chemical and physical conditions. Algal cells appear to markedly concentrate metals from solution, even at concentrations of these metals in the medium which do not apparently inhibit cell division. Bioassays provide the only direct method for assessing the biological availability of metals in solution. Algae isolated from metal-polluted lakes appear to have evolved specific metal tolerances. These "tolerant" algae actually accumulate more of the metals concerned than do their "nontolerant" relatives. Correlations between fish toxicity tests and algal bioassays may allow the relatively expensive fish testing schemes to be replaced by simple and cheaper algal bioassays.

Abstract  Nano-silver (Ag) with antimicrobial activity is by far the most commercialized nano-compound. The hazards associated with human exposure to nanosized-silver should be investigated to facilitate the risk assessment process. Recent studies have shown that inflammatory, oxidative, genotoxic, and cytotoxic consequences are associated with silver particulate exposure, and are inherently linked. In the present study, the cytotoxicity and genotoxicity of nano-silver were investigated using the dye exclusion assay, the comet assay, and the mouse lymphoma thymidine kinase (tk+/−) gene mutation assay (MLA). IC20 values of nano-silver in L5178Y cells were determined the concentration of 3,769.53 μg/mL and 1,796.88 μg/mL with and without S-9, respectively. And in BEAS-2B cell, IC20 values were calculated to 1,171.88 μg/mL and 761.72 μg/mL with and without S-9, respectively. From these results, nano-silver was more cytotoxic in absence of S-9 metabolic activation system and at the BEAS-2B cells. In the comet assay, L5178Y cells and BEAS-2B cells were treated with nano-silver which significantly increased >2-fold tail moment with and without S-9. However, the mutant frequencies in the nano-silver treated L5178Y cells were slightly increased but not significant compared to the vehicle controls with and without S-9. The results of this study indicate that nano-silver can cause primary DNA damage and cytotoxicity but not mutagenicity in cultured mammalian cells.

Abstract  Silver nanoparticles (AgNPs) have emerged as an important class of nanomaterials and are currently used in a wide range of industrial and commercial applications. This has caused increasing concern about their effects on the environment and to human health. Using Japanese medaka (Oryzias latipes) at early-life stages as experimental models, the developmental toxicity of silver nanoparticles was investigated following exposure to 100–1000 μg/L homogeneously dispersed AgNPs for 70 days, and developmental endpoints were evaluated by microscopy during embryonic, larval and juvenile stages of development in medaka. Meanwhile, histopathological changes in the larval eye were evaluated. Retarded development and reduced pigmentation were observed in the treated embryos by AgNPs at high concentrations (≥400 μg/L). Maximum width of the optic tectum, as an indicator of midbrain development, decreased significantly in a dose-related manner. Furthermore, silver nanoparticles exposure at all concentrations induced a variety of morphological malformations such as edema, spinal abnormalities, finfold abnormalities, heart malformations and eye defects. Histopathological observations also confirmed the occurrence of abnormal eye development induced by AgNPs. The data showed non-linear or U-shaped dose–response patterns for growth retardation at 5 days of postfertilization, as well as the incidence of abnormalities. Preliminary results suggested that the developmental process of medaka may be affected by exposure to silver nanoparticles. Morphological abnormalities in early-life stages of medaka showed the potential developmental toxicities of silver nanoparticles. Further research should be focused on the mechanisms of developmental toxicity in fish exposed to silver nanoparticles.

Abstract  The eco- and genotoxicity of silver nanoparticles (AgNPs) was investigated in the fourth instar larvae of the aquatic midge, Chironomus riparius. AgNPs did not have acute toxicity in C. riparius, but did exhibited chronic toxicity on development (pupation and emergence failure) and reproduction. Genotoxicity also occurred in AgNPs exposed C. riparius. Differential Display PCR (DD-PCR), based on the Annealing Control Primer (ACP) technique, was conducted to investigate the underlying toxic mechanism, which identified altered gene expression in C. riparius after treatment with AgNPs. The possible toxicity mechanism of AgNPs in C. riparius involves the down regulation of the ribosomal protein gene (CrL15) affecting the ribosomal assembly and consequently, protein synthesis. Up regulation of the gonadotrophin releasing hormone gene (CrGnRH1) might lead to the activation of gonadotrophin releasing hormone mediated signal transduction pathways and reproductive failure. Up regulation of the Balbiani ring protein gene (CrBR2.2) may be an indication of the organism's protection mechanism against the AgNPs. The overall results suggest that the toxicity of AgNPs towards aquatic organisms should be thoroughly investigated to allow for their safe use, as they seem to exhibit important toxicity towards C. riparius.

Abstract  The rapid development and potential release of engineered nanoparticles (ENPs) have raised considerable concerns due to the unique properties of nanomaterials. An important aspect of the risk assessment of ENPs is to understand the interactions of ENPs with plants, an essential base component of all ecosystems. The impact of ENPs on plant varies, depending on the composition, concentration, size and other important physical chemical properties of ENPs and plant species. Both enhancive and inhibitive effects of ENPs on plant growth at different developmental stages have been documented. ENPs could be potentially taken up by plant roots and transported to shoots through vascular systems depending upon the composition, shape, size of ENPs and plant anatomy. Despite the insights gained through many previous studies, many questions remain concerning the fate and behavior of ENPs in plant systems such as the role of surface area or surface activity of ENPs on phytotoxicity, the potential route of entrance to plant vascular tissues and the role of plant cell walls in internalization of ENPs. This article reviewed the current knowledge on the phytotoxicity and interactions of ENPs with plants at seedling and cellular levels and discussed the information gap and some immediate research needs to further our knowledge on this topic.

Abstract  Nanoparticles are small scale substances (<100 nm) used in biomedical applications, electronics, and energy production. Increased exposure to nanoparticles being produced in large-scale industry facilities elicits concerns for the toxicity of certain classes of nanoparticles. This study evaluated the effects of silver-25 nm (Ag-25) nanoparticles on gene expression in different regions of the mouse brain. Adult-male C57BL/6N mice were administered (i.p.) 100mg/kg, 500 mg/kg or 1,000 mg/kg Ag-25 and sacrificed after 24h. Regions from the brain were rapidly removed and dissected into caudate nucleus, frontal cortex and hippocampus. Total RNA was isolated from each of the three brain regions collected and real-time RT-PCR analysis was performed using Mouse Oxidative Stress and Antioxidant Defense Arrays. Array data revealed the expression of genes varied in the caudate nucleus, frontal cortex and hippocampus of mice when treated with Ag-25. The data suggest that Ag-25 nanoparticles may produce neurotoxicity by generating free radical-induced oxidative stress and by altering gene expression, producing apoptosis and neurotoxicity.

Abstract  The impact of capping agents and environmental conditions (pH, ionic strength, and background electrolytes) on surface charge and aggregation potential of silver nanoparticles (AgNPs) suspensions were investigated. Capping agents are chemicals used in the synthesis of nanoparticles to prevent aggregation. The AgNPs examined in the study were as follows: (a) uncoated AgNPs (H(2)-AgNPs), (b) electrostatically stabilized (citrate and NaBH(4)-AgNPs), (c) sterically stabilized (polyvinylpyrrolidone (PVP)-AgNPs), and (d) electrosterically stabilized (branched polyethyleneimine (BPEI)-AgNPs)). The uncoated (H(2)-AgNPs), the citrate, and NaBH(4)-coated AgNPs aggregated at higher ionic strengths (100 mM NaNO(3)) and/or acidic pH (3.0). For these three nanomaterials, chloride (Cl(-), 10 mM), as a background electrolyte, resulted in a minimal change in the hydrodynamic diameter even at low pH (3.0). This was limited by the presence of residual silver ions, which resulted in the formation of stable negatively charged AgCl colloids. Furthermore, the presence of Ca(2+) (10 mM) resulted in aggregation of the three previously identified AgNPs regardless of the pH. As for PVP coated AgNPs, the ionic strength, pH and electrolyte type had no impact on the aggregation of the sterically stabilized AgNPs. The surface charge and aggregation of the BPEI coated AgNPs varied according to the solution pH.

Abstract  The purpose of this study was to investigate the effect of surface coating on the toxicity of silver nanoparticles (Ag NPs) soil. Earthworms (Eisenia fetida) were exposed to AgNO(3) and Ag NPs with similar size ranges coated with either polyvinylpyrrolidone (hydrophilic) or oleic acid (amphiphilic) during a standard sub-chronic reproduction toxicity test. No significant effects on growth or mortality were observed within any of the test treatments. Significant decreases in reproduction were seen in earthworms exposed to AgNO3, (94.21 mg kg(-1)) as well as earthworms exposed to Ag NPs with either coating (727.6 mg kg(-1) for oleic acid and 773.3 mg kg(-1) for polyvinylpyrrolidone). The concentrations of Ag NPs at which effects were observed are much higher than predicted concentrations of Ag NPs in sewage sludge amended soils; however, the concentrations at which adverse effects of AgNO(3) were observed are similar to the highest concentrations of Ag presently observed in sewage sludge in the United States. Earthworms accumulated Ag in a concentration-dependent manner from all Ag sources, with more Ag accumulating in tissues from AgNO(3) compared to earthorms exposed to equivalent concentrations of Ag NPs. No differences were observed in Ag accumulation or toxicity between earthworms exposed to Ag NPs with polyvinylpyrrolidone or oleic acid coatings.

Abstract  Silver nanoparticles (Ag NPs) have recently received much attention for their possible applications in biotechnology and life sciences. Ag NPs are of interest to defense and engineering programs for new material applications as well as for commercial purposes as an antimicrobial. However, little is known about the genotoxicity of Ag NPs following exposure to mammalian cells. This study was undertaken to examine the DNA damage response to polysaccharide surface functionalized (coated) and non-functionalized (uncoated) Ag NPs in two types of mammalian cells; mouse embryonic stem (mES) cells and mouse embryonic fibroblasts (MEF). Both types of Ag NPs up-regulated the cell cycle checkpoint protein p53 and DNA damage repair proteins Rad51 and phosphorylated-H2AX expression. Furthermore both of them induced cell death as measured by the annexin V protein expression and MTT assay. Our observations also suggested that the different surface chemistry of Ag NPs induce different DNA damage response: coated Ag NPs exhibited more severe damage than uncoated Ag NPs. The results suggest that polysaccharide coated particles are more individually distributed while agglomeration of the uncoated particles limits the surface area availability and access to membrane bound organelles.

Abstract  Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 microg/mL and 100 microg/mL SNP, respectively, although with minor decrease in confluence. IC(50) values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 microg/mL and 449 microg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC(50) SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (i.e. 30 microg/mL and 225 microg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (approximately 1.2 fold) and depletion of lipid peroxidation (approximately 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ( approximately 1.4 fold) and GSH ( approximately 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 microg/mL in primary fibroblasts, 12.5 microg/mL in primary liver cells) than the necrotic concentration (100 microg/mL in primary fibroblasts, 500 microg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC(50) SNP (resulting in apoptosis) and 2 x IC(50)) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (approximately 20 microg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.

Abstract  The environmental genotoxic behavior of silver nanoparticles (nanoAg) combined with the detergent cetylpyridine bromide (CPB) was examined in vitro. The experimental results showed that the genotoxicity of nanoAg itself is weak, but nanoAg shows obvious genotoxicity after combined with CPB. The combined materials have a strong coeffect on calf thymus DNA (ctDNA) at a concentration of 3.3 × 10−6 g mL−1 nanoAg and 6.0 × 10−6 mol L−1 CPB. After the addition of ctDNA to the nanoAg–CPB system, the particles are scattered and the diameter decreases, which indirectly reveal that nanoAg–CPB has genotoxicity.

Abstract  Silver nanoparticles (nano-Ag) are potent and broad-spectrum antimicrobial agents. In this study, spherical nano-Ag (average diameter = 9.3 nm) particles were synthesized using a borohydride reduction method and the mode of their antibacterial action against E. coli was investigated by proteomic approaches (2-DE and MS identification), conducted in parallel to analyses involving solutions of Ag(+) ions. The proteomic data revealed that a short exposure of E. coli cells to antibacterial concentrations of nano-Ag resulted in an accumulation of envelope protein precursors, indicative of the dissipation of proton motive force. Consistent with these proteomic findings, nano-Ag were shown to destabilize the outer membrane, collapse the plasma membrane potential and deplete the levels of intracellular ATP. The mode of action of nano-Ag was also found to be similar to that of Ag(+) ions (e.g., Dibrov, P. et al, Antimicrob. Agents Chemother. 2002, 46, 2668-2670); however, the effective concentrations of nano-Ag and Ag(+) ions were at nanomolar and micromolar levels, respectively. Nano-Ag appear to be an efficient physicochemical system conferring antimicrobial silver activities.

Abstract  Nanotechnology is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Among the most promising nanomaterials with antibacterial properties are metallic nanoparticles, which exhibit increased chemical activity due to their large surface to volume ratios and crystallographic surface structure. The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. The present work studies the effect of silver nanoparticles in the range of 1-100 nm on Gram-negative bacteria using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). Our results indicate that the bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of [?]1-10 nm.

Abstract  In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag+ (in the form of AgNO3). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.

Abstract  The antimicrobial activity of silver nanoparticles against E. coli was investigated as a model for Gram-negative bacteria. Bacteriological tests were performed in Luria?Bertani (LB) medium on solid agar plates and in liquid systems supplemented with different concentrations of nanosized silver particles. These particles were shown to be an effective bactericide. Scanning and transmission electron microscopy (SEM and TEM) were used to study the biocidal action of this nanoscale material. The results confirmed that the treated E. coli cells were damaged, showing formation of ?pits? in the cell wall of the bacteria, while the silver nanoparticles were found to accumulate in the bacterial membrane. A membrane with such a morphology exhibits a significant increase in permeability, resulting in death of the cell. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.

Abstract  The ATSDR toxicological profile succinctly characterizes the toxicologic and adverse health effects information for the hazardous substance described here. Each peer-reviewed profile identifies and reviews the key literature that describes a hazardous substance's toxicologic properties. Other pertinent literature is also presented, but is described in less detail than the key studies.