E56 - Nanotechnology

SIGNIFICANCE AND USE
5.1 Liposomal formulations for the treatment of cancer and other diseases are the most common form of nanotechnology-enabled drug products submitted for market approval and in clinical application at the present time. The accurate characterization of their physical-chemical properties is critical to support the development and assessment of such products (2). In particular, size, size distribution, shape, and physical stability are key properties (among others) that have been widely identified as critical quality attributes (CQAs) for liposomal drug products.  
5.1.1 Asymmetrical-flow field-flow fractionation (AF4) is a chromatographic-like technique that uses hydrodynamic forces to gently separate analytes into their component populations according to size and diffusivity (3 and 4). The fractionated sample then passes through one or more online detectors chosen specifically for the application requirements. The combination of low-shear separation, tolerance for complex matrices, and exceptionally broad size range make AF4 a technique of choice for application to nanotechnology-enabled drug products such as liposomes (13, 5-9).  
5.1.2 Multi-detector AF4 is suitable for research and development, manufacturing quality control, product stability/shelf-life testing and regulatory assessments.  
5.1.3 There are multiple assumptions inherent in the application of MD-AF4, including the appropriateness of models used to interpret online light scattering data and the compatibility of the analyte and matrix with the membrane that forms the surface of the accumulation wall in the fractionation channel. Other assumptions are detector or analyte specific.  
5.2 Chemical components of the mobile phase must not induce agglomeration of liposomes or otherwise significantly alter their physical properties.  
5.3 Discretion should be used in the interpretation of size data obtained by different scattering detectors using different scattering models and modes of analysis.  
5.4 ...
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1.1 This test method describes a measurement procedure to reproducibly separate component size populations present within liposomal drug formulations and to characterize their associated size and size distribution. The method can also yield information on the shape and physical stability of the liposomes and is applicable to measurements in the presence of serum proteins. Fractions can be collected for off-line analysis using various techniques not specified in this test method.  
1.2 This test method applies to uni-lamellar and multi-lamellar liposomes that are designed for drug delivery and which are dispersed in a native solution that is aqueous in nature. The method is generally applicable over a particle size range (radius) of approximately 10 nm to 250 nm, and for injected lipid mass from 20 µg to 200 µg.  
1.3 This test method is based on the multi-detector asymmetrical-flow field-flow fractionation (MD-AF4) technique as configured on a typical commercial instrument platform with online detectors such as multi-angle (static) light scattering (MALS), dynamic light scattering (DLS), ultraviolet-visible (UV-Vis) absorbance, and differential refractive index (dRI) (1).2  
1.4 This method does not address liposome composition. Refer to Test Methods E3297, E3323, or E3324 for lipid quantification.  
1.5 Units—The values stated in SI units are to be regarded as standard. Where appropriate, cgs units are given in addition to SI.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of In...

SIGNIFICANCE AND USE
5.1 As AFM measurement technology has matured and proliferated, the technique has been widely adopted by the nanotechnology research and development community to the extent that it is now considered an indispensible tool for visualizing and quantifying structures on the nanoscale. Whether used as a stand-alone method or to complement other dimensional measurement methods, AFM is now a firmly established component of the nanoparticle measurement tool box. International standards for AFM-based determination of nanoparticle size are nonexistent as of the drafting of this guide. Therefore, this standard aims to provide practical and metrological guidance for the application of AFM to measure the size of substrate-supported nanoparticles based on maximum displacement as the probe is rastered across the particle surface to create a line profile.
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1.1 The purpose of this document is to provide guidance on the quantitative application of atomic force microscopy (AFM) to determine the size of nanoparticles2 deposited in dry form on flat substrates using height (z-displacement) measurement. Unlike electron microscopy, which provides a two-dimensional projection or a two-dimensional image of a sample, AFM provides a three-dimensional surface profile. While the lateral dimensions are influenced by the shape of the probe, displacement measurements can provide the height of nanoparticles with a high degree of accuracy and precision. If the particles are assumed to be spherical, the height measurement corresponds to the diameter of the particle. In this guide, procedures are described for dispersing gold nanoparticles on various surfaces such that they are suitable for imaging and height measurement via intermittent contact mode AFM. Generic procedures for AFM calibration and operation to make such measurements are then discussed. Finally, procedures for data analysis and reporting are addressed. The nanoparticles used to exemplify these procedures are National Institute of Standards and Technology (NIST) reference materials containing citrate-stabilized negatively charged gold nanoparticles in an aqueous solution.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.
FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEM
Note 1: Both images are shown to the same scale; scale bar is 200 nm.  
5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same liposomal preparation is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.  
5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in a cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface o...
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1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 A tiered strategy for characterization of nanoparticle properties is necessary to draw meaningful conclusions concerning dose-response relationships observed during inhalation toxicology experiments. This tiered strategy includes characterization of nanoparticles as produced (that is, measured as the bulk material sold by the supplier) and as administered (that is, measured at the point of delivery to a test subject) (Oberdorster et al. (6)).  
5.2 Test Methods B922 and C1274 and ISO 9277 and ISO 18757 exist for determination of the as produced surface area of bulk metal and metal oxide powders. During the delivery of nanoparticles in inhalation exposure chambers, the material properties may undergo change and therefore have properties that differ from the material as produced. This test method describes the determination of the as administered surface area of airborne metal oxide nanoparticles in inhalation exposure chambers for inhalation toxicology studies.
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1.1 This test method covers determination of surface area of airborne metal oxide nanoparticles in inhalation exposure chambers for inhalation toxicology studies. Surface area may be measured by gas adsorption methods using adsorbates such as nitrogen, krypton, and argon (Brunauer et al. (1),2 Anderson (2), Gregg and Sing (3)) or by ion attachment and mobility-based methods (Ku and Maynard (4)). This test method is specific to the measurement of surface area by gas adsorption by krypton gas adsorption. The test method permits the use of any modern commercial krypton adsorption instruments but strictly defines the sample collection, outgassing, and analysis procedures for metal and metal oxide nanoparticles. Use of krypton is required due to the low overall surface area of particle-laden samples and the need to accurately measure the background surface area of the filter used for sample collection. Instrument-reported values of surface area based on the multipoint Brunauer, Emmett and Teller (BET) equation (Brunauer et al. (1), Anderson (2), Gregg and Sing (3)) are used to calculate surface area of airborne nanoparticles collected on a filter.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. State all numerical values in terms of SI units unless specific instrumentation software reports surface area using alternate units.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 Natural and manufactured textiles fibers can be treated with chemicals to provide enhanced antimicrobial (fungi, bacteria, viruses) properties. In some cases, silver nanomaterials may be used to treat textile fibers (1).6 Silver nanomaterials are used to treat a wide array of consumer textile products, including, but not limited to, various clothing; primary garments (shirts, pants), outer wear (gloves, jackets), inner wear (socks and underwear), children’s clothing (sleepwear); children’s plush toys; bath towels and bedding (sheets, pillows); and medical devices (for example, wound dressings and face masks) (2).  
4.2 There are many different chemical and physical forms of silver that are used to treat textiles and an overview of this topic is provided in Appendix X1.  
4.3 Several applicable techniques for detection and characterization of silver are listed and described in Appendix X2 so that users of this guide may understand the suitability of a particular technique for their specific textile and silver measurement need.  
4.4 There are many different reasons to assay for silver nanomaterials in a textile at any point in a product’s life cycle. For example, a producer may want to verify that a textile meets their internal quality control specifications or a regulator may want to understand the properties of silver nanomaterials used to make a consumer textile product under their jurisdiction or what quantity of silver nanomaterial is potentially available for release from the treated textile during the washing process or during product use. Regardless of the specific reason, a structured approach to detect and characterize silver nanomaterials present in a textile will facilitate measurements and data comparison. Detection and characterterization of silver in textiles is one component of an overall risk assessment.  
4.5 The approach presented in this guide (see Fig. 1) consists of three sequential tiers: obtain a textile sample (Section 7), detection o...
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1.1 This guide covers the use of a tiered approach for detection and characterization of silver nanomaterials in consumer textile products, which can include some medical devices (for example, wound dressings or face masks), made of any combination of natural or manufactured fibers.  
1.2 This guide covers, but is not limited to, fabrics and parts (for example, thread, batting) used during the manufacture of textiles and production of consumer textile products that may contain silver-based nanomaterials. It does not apply to analysis of silver nanomaterials in non-consumer textile product matrices nor does it cover thin film silver coatings with only one dimension in the nanoscale.  
1.3 This guide is intended to serve as a resource for manufacturers, producers, analysts, policymakers, regulators, and others with an interest in textiles.  
1.4 This guide is presented in the specific context of measurement of silver nanomaterials; however, the structured approach described herein is applicable to other nanomaterials in consumer textile products, including some medical devices.  
1.5 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Lipid composition in a liposomal formulation is an important aspect during synthesis of liposomes, which determines stability, surface characteristics, drug encapsulation, and drug release capabilities. The cholesterol component plays a key role in controlled drug release by adding stability to the liposome. A small variation in the lipid composition can significantly alter the parameters mentioned above (15).  
5.2 Variation in the lipid composition in the liposomal formulation may influence the safety and efficacy of the product. Therefore, chemical composition of the liposomes shall be determined.  
5.3 The pharmaceutical industry and regulatory agencies require QC, QA, specifications, thorough characterization, and quantification of lipid components (16, 17).  
5.4 This test method can be used to ascertain variations in the lipid component profiling of various liposomal formulations. However, this test method does not intend to identify chemical degradation products (18).  
5.5 Analyzing the stability of analytes and their chemical degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method (18, 19).
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1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal formulations as major components.  
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and evaporative light-scattering detection (ELSD) to quantify the individual components.  
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-phosphatidylcholine (HSPC).  
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.  
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method development and method validation, sample analysis, and data reporting.  
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 2 µg/g to 4 µg/g and 7 µg/g to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC are 10 µg/g to 165 µg/g, 10 µg/g to 300 µg/g, and 10 µg/g to 200 µg/g, respectively.  
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in Practice D6026.  
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.  
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 NTA is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region. This guide describes the NTA technique for direct visualization and measurement of Brownian motion, generally applicable in the particle size range from several nanometers until the onset of sedimentation in the sample. The NTA technique is usually applied to dilute suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from size diameters determined by other techniques and users of the NTA technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques (see 8.7). Notwithstanding the preceding sentence, the technique is routinely applied in industry and academia as both a research and development tool and as a QC method for the characterization of submicron systems.
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1.1 This guide deals with the measurement of particle size distribution of suspended particles, from ~10 nm to the onset of sedimentation, sample dependent, using the nanoparticle tracking analysis (NTA) technique. It does not provide a complete measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that should be followed for good practice, along with potential pitfalls.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This test method is one of a series of tests listed in Practice F748 and ISO 10993-4 to assess the biocompatibility of materials contacting blood in medical applications.  
5.2 This test method is similar to Practice F756 but modified to accommodate nanoparticulate materials.
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1.1 This test method covers assessing the effect of nanoparticulate materials on the integrity of red blood cells.  
1.2 This test method uses diluted whole blood incubated with nanoparticulate material and the hemoglobin released from damaged red blood cells is determined.  
1.3 This test method is similar to Practice F756 with the volumes reduced to accommodate nanoparticulate material.  
1.4 This test method is part of the in-vitro preclinical characterization and is important for nanoparticulate material that will contact the blood in medical applications.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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1.1 This guide deals with the measurement of mobility and zeta potential in systems containing biological material such as proteins, DNA, liposomes and other similar organic materials that possess particle sizes in the nanometer scale (  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Mean particle diameters defined according to the Moment-Ratio (M-R) system are derived from ratios between two moments of a particle size distribution.
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1.1 The purpose of this practice is to present procedures for calculating mean sizes and standard deviations of size distributions given as histogram data (see Practice E1617). The particle size is assumed to be the diameter of an equivalent sphere, for example, equivalent (area/surface/volume/perimeter) diameter.  
1.2 The mean sizes/diameters are defined according to the Moment-Ratio (M-R) definition system.2,3,4  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This test method is designed to evaluate nanomaterial capacity to induce nitric oxide production by macrophages.  
5.2 Activated macrophages generate large quantities of NO. NO generated from activated macrophages is a cytostatic/cytotoxic agent (3-6).  
5.3 The production of NO in excessive amounts leads to the generation of peroxynitrite by its spontaneous reaction with superoxide. Peroxynitrite causes tissue injury through its capability to damage lipids, proteins, and DNA (2).  
5.4 NO is a proinflammatory mediator and it is an important marker for activation of inflammation (5, 6).  
5.5 Testing the capacity of a nanomaterial to induce NO production in vitro helps in predicting the nanomaterial’s biocompatibility through anticipating and understanding the potential problems that might be encountered during its in vivo administration.
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1.1 This test method delivers a protocol for a quantitative measure of nitrite (NO2–), a stable end-product of nitric oxide (NO), in cell culture medium due to exposure to nanomaterial(s).  
1.2 NO has a critical role in several pathological conditions in addition to its role in many physiological processes.  
1.3 This test method uses murine macrophage cell line RAW 264.7 as an in vitro model.  
1.4 The nitrite is measured in the cell culture medium by a colorimetric analysis using Griess reagent as shown in Fig. 1.
FIG. 1 Summary of Nitric Oxide Production Assay  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Stem cells of hematopoietic origin are pluripotential and may be particularly sensitive to the effects of stimulation by nanoparticulate materials.  
5.2 The effect of particles on macrophage responses has an extensive history and can be assessed by Practice F1903. The test method described here will assess the effect on stem cells which can be progenitor cells to the macrophage line.
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1.1 This test method provides a protocol for quantitative analysis of the effect of nanoparticulate materials in physiologic solution (isotonic, pH 7.2 ± 0.2) on granulocyte-macrophage colony-forming units (CFU-GM).  
1.2 CFU-GM reflects the number of viable bone marrow cells which differentiate into granulocytes and macrophages. A decrease in CFU-GM count is indicative of a test substance’s toxicity to bone marrow and is commonly used for the identification of drug products with myelosuppressive properties, a form of immunosuppression.  
1.3 This test method employs murine bone marrow hematopoietic stem cells which proliferate and differentiate to form discrete cell clusters or colonies which are counted.  
1.4 This test method is part of the in vitro preclinical characterization cascade for nanoparticulate materials for systemic administration in medical applications.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Assessing the propensity of a nanomaterial to cause cytotoxicity to the cells of a target organ can assist in preclinical development.  
5.2 The standard historical cytotoxicity testing of materials and extracts of materials has used fibroblasts and is well documented in Practice F813, Test Method F895, and ISO 10993-5. The use of macrophages and micron size particles has also provided information on cytotoxicity and stimulation using Practice F1903.  
5.3 This test method adds to the cytotoxicity test protocols by using target organ cells. Two quantitative assays measuring LDH leakage and MTT reduction are used to estimate cytotoxicity.  
5.4 This test method may not be predictive of events occurring in all types of nanomaterial applications, and the user is cautioned to consider the appropriateness of the test for various types of nanomaterial and their applications. This procedure should only be used to compare the cytoxicity of a series of related nanomaterials. Meaningful comparison of unrelated nanomaterials is not possible without additional characterization of physicochemical properties of each individual nanomaterial in the assay matrix.
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1.1 This test method provides a methodology to assess the cytotoxicity of suspensions of nanoparticulate materials in porcine proximal tubule cells (LLC-PK1) and human hepatocarcinoma cells (Hep G2), which represent potential target organs following systemic administration.  
1.2 This test method is part of an in vitro preclinical characterization cascade.  
1.3 This test method consists of a protocol utilizing two methods for estimation of cytotoxicity, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide establishes, at the undergraduate college level, the basic education structure for understanding the unique properties and applications of nanoscale materials as compared to bulk properties and applications of macroscale materials. It helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research and can be used by organizations developing or carrying out education programs for the nanotechnology workforce.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently.  
5.3 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of material properties and the effects of size, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to maintain an up-to-date understanding of the technology and have sufficient base education to enable the synthesis of emerging or evolving safety procedures and practices.  
5.5 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
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1.1 This guide provides a framework for a basic workforce education in material properties at the nanoscale, to be taught at an undergraduate college level. This education should be broad to prepare an individual to serve within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This guide may be used to develop or evaluate an education program for unique material properties and their applications in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this guide, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the techniques, materials, and concepts needed for material properties and applications. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide, at the undergraduate college level, a basic educational structure in the synthesis and processing of nanoscale materials to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently.  
5.3 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of topics related to the infrastructure required for advanced research and manufacturing, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to maintain an up-to-date understanding of the technology and have sufficient base education to enable the synthesis of emerging or evolving safety procedures and practices.  
5.5 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
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1.1 This guide provides a framework for a basic workforce education in materials synthesis and processing at the nanoscale, to be taught at an undergraduate college level. This education should be broad to prepare an individual to serve within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This guide may be used to develop or evaluate an education program for synthesis and processing applications in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this practice, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the techniques, materials, and concepts needed for materials synthesis and processing at the nanoscale. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide, at the undergraduate college level, a basic educational structure in the infrastructure aspects of nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently.  
5.3 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of topics related to the infrastructure required for advanced research and manufacturing, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to maintain an up-to-date understanding of the technology and have sufficient base education to enable the synthesis of emerging or evolving safety procedures and practices.  
5.5 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
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1.1 This document provides guidelines for basic workforce education in the infrastructure topics related to nanotechnology to be taught at an undergraduate college level. This education should be broad to prepare an individual to work within one of the many areas in nanotechnology research, development, or manufacturing. The individual so educated may be involved in material handling, manufacture, distribution, storage, use, or disposal of nanoscale materials.  
1.2 This guide may be used to develop or evaluate an education program for the infrastructure used in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this guide, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the methods and concepts pertaining to the infrastructure for nanotechnology. It may not cover knowledge and skill objectives applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Liposomes are vesicles of nanoscale dimensions, composed of lipid bilayers, which are used for various diagnostic and therapeutic applications (9). The growing interest in liposomal formulations in the delivery of various drugs, antisense oligonucleotides, cloned genes, or recombinant proteins by the biopharmaceutical industry, warrants QC and thorough characterization of the constituent lipids. Lipid structure, composition, and concentration are key attributes in determining the quality and efficacy of a liposomal drug product as they influence the stability of liposomes, drug loading, release kinetics, biodistribution, and pharmacokinetic properties (9). Cholesterol modulates the lipid membrane fluidity, elasticity, and permeability; hence, it plays a key role in controlled drug release and increased stability of the liposome (10).  
5.2 This test method provides a rapid and reliable protocol for the determination of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations using UHPLC-TQMS. Assessment of the stability of the analytes in terms of their degradation profiles is not included in this test method (11). This test method will benefit the biopharmaceutical industry in ascertaining quality assessment of liposomal formulations and monitoring batch-to-batch consistency for large-scale production, thereby facilitating safe and efficient drug development and regulatory review.  
5.3 UHPLC-MS/MS measurements are analytically more sensitive and specific for lipid analysis compared to other contemporary techniques using universal detectors, such as a charged aerosol or an evaporative light-scattering detector. For liposomes, MS/MS has further advantages over ultraviolet detectors, as lipids lack chromophores for detection. In this test method, TQMS has been used as the MS/MS technique of choice because of its high selectivity, sensitivity, S/N, accuracy, and broad linear range of quantitation, thereby allowing reproducible quantitation of the analytes,...
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1.1 This test method describes the determination of lipid components in liposomal formulations, which includes sample solubilization in methanol followed by separation of the analytes using ultra-high-performance liquid chromatography (UHPLC) and detection with tandem mass-spectrometry (MS/MS). This test method adheres to multiple reaction monitoring (MRM) mass spectrometry on a triple quadrupole mass spectrometer (TQMS).  
1.2 This test method is specific for liposomal formulations containing cholesterol, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE- PEG 2000), and hydrogenated (soy) L-α-phosphatidylcholine (HSPC).  
1.3 This test method is applicable to report the absolute concentrations of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations. Assessment of the stability of the analytes in terms of their degradation as a result of oxidation or hydrolysis is beyond the scope of this test method.  
1.4 This test method includes calibration and standardization, sample preparation, UHPLC-TQMS instrumentation, potential interferences, method validation with acceptance criteria, sample analysis, and data reporting.  
1.5 The detection limits for cholesterol, DSPE-PEG 2000, and HSPC using this test method are 5.3, 0.5, and 0.5 ng/g, respectively. In addition, the quantitation limits for cholesterol, DSPE-PEG 2000, and HSPC are 10.6, 0.8, and 0.5 ng/g, respectively.  
1.6 This test method is intended for concentration ranges of 8-1600 ng/g for cholesterol, and of 2-400 ng/g for DSPE-PEG 2000 and HSPC.  
1.7 All observed and calculated values shall conform to the guidelines for significant digits and rounding as established in Practice D6026.  
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.  
1.9 Th...

SIGNIFICANCE AND USE
5.1 The growing interest in liposomal formulations in the pharmaceutical industry requires QC and thorough characterization and quantification of lipids that form liposomes (6). Lipid composition has proven to be a critical attribute of the liposomal formulation; it directly influences the stability of the formulation, drug loading, performance, size, and surface characteristics of the liposome. Cholesterol plays a key role in controlled drug release by adding stability to the liposome (7). Significant variation in the lipid composition and ratio of the components will influence the safety, biodistribution, drug efficacy, and drug release kinetics of the liposomal formulation (8-11).  
5.2 This test method is a fast and reliable procedure for the quantification of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations using HPLC-CAD.  
5.3 This test method can be used for QC and QA and to ascertain variations in component profiling of liposomal formulations.
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1.1 This test method is for the separation of lipids in liposomal formulations through high performance liquid chromatography (HPLC) and their quantitation using a mass-flow sensitive charged aerosol detector (CAD).  
1.2 This test method is specifically for liposomal formulations containing cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000) and hydrogenated soy L-α-phosphatidylcholine (HSPC).  
1.3 This test method is applicable to report the absolute concentrations and ratio of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations. Assessment of the stability of the analytes in terms of their degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method.  
1.4 This test method includes calibration standards preparation, sample preparation, method validation, and sample analysis. This method also contains specifications for instrumentation and the chromatography experimental procedure.  
1.5 The detection limit and quantitation limit for the analytes in this test method is in the range of 0.1–2.0 µg/g and 1.0–5.0 μg/g respectively. The analytical measurement range for cholesterol, DSPE-PEG 2000, and HSPC is 5–300 µg/g.  
1.6 All observed and calculated values shall conform to the guidelines for significant digits and rounding as established in Practice D6026.  
1.7 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Silver may be used to treat consumer textile products to provide enhanced antimicrobial (fungi, bacteria, viruses) properties (3, 4). At any point in a textile product’s lifecycle, there may be a need to measure the amount of silver present. This standard prescribes a test method based on ICP-OES or ICP-MS analysis that manufacturers, producers, analysts, policymakers, regulators, and others may use for measurement of total silver in textiles. As described in Guide E3025, determination of total silver in a consumer textile product is one component of a tiered approach to determine if silver is present, possibly as nanomaterial(s) (one or more external dimensions in the nanoscale), prior to measuring the form and dimension of the Ag that is found. ICP-OES or ICP-MS analysis alone is not sufficient to determine whether a textile contains silver nanomaterial(s).
Note 4: There are many different chemical and physical forms of silver that are used to treat textiles and an overview of this topic is provided in Guide E3025.  
5.2 As described in Guide E3025, the amount of silver in a textile can decrease over time as silver metal and silver compounds can react with oxygen and other oxidation-reduction (redox) active agents present in the environment to form soluble ionic species which are released by contact with moisture (for example, from ambient humidity, washing, body sweat, rain, or other sources). Hence, if silver is measured in a textile, the result may only be indicative of that moment in the article’s life cycle and great care is necessary in drawing temporal inferences from the results.  
5.3 If silver is measured by ICP-OES or ICP-MS analysis, additional analyses are needed to elucidate the form of silver in the textile specimen. This step is necessary because ICP-OES or ICP-MS results are for total silver independent of chemical and physical form and textiles may be treated with silver in sizes that range from the nanoscale (for example, salt nanopartic...
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1.1 This test method covers the use of inductively coupled plasma–optical emission spectrometry (ICP-OES) and inductively coupled plasma–mass spectrometry (ICP-MS) analyses for determination of the mass fraction of total silver in consumer textile products made of any combination of natural or manufactured fibers. Either ICP-OES or ICP-MS analysis is recommended as a first step to test for and quantify silver in a textile and results can be used to inform subsequent, more detailed analyses as part of the tiered approach described in Guide E3025 to determine if a textile contains silver nanomaterial(s).  
1.2 This test method prescribes acid digestion to prepare test sample solutions from samples of textiles utilizing an appropriate internal standard followed by external calibration and analysis with either ICP-OES or ICP-MS to quantify total silver.  
1.3 This test method is believed to provide quantitative results for textiles made of fibers of rayon, cotton, polyester, and lycra that contain metallic silver (see Section 17). It is the analyst’s responsibility to establish the efficacy (ability to achieve the planned and desired analytical result) of this test method for other textile matrices and forms of silver.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurements are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organiz...

SIGNIFICANCE AND USE
5.1 The information and recommendations in this guide are relevant for imaging and identifying ENMs in cells and other biological (for example, fixed tissues, whole plants) and nonbiological (for example, drug formulations, filter media, soil, and wastewater) matrices after appropriate sample preparation procedures have been performed (3-5). DFM/HSI is a recently developed analytical tool; however, the relative simplicity of sample preparation combined with the potential to acquire high-contrast ENM images and high-content ENM spectral responses facilitates the increasing use of the tool for diverse applications in drug delivery, toxicology, environmental science, biology, and medicine.  
5.2 Verification of the uptake and spatial distribution of ENMs in cells, for example, is necessary for evaluating and understanding the biological effects of ENMs on living systems. Similarly, the closeness of the spatial distribution of ENMs in complex drug formulations can be an important criterion in establishing physicochemical similarity between formulations (6). Complex products are described in the most recent version of the Generic Drug User Fee Act (GDUFA) reauthorization commitment letter: (7). This guide covers the criteria and general considerations for performing DFM/HSI analyses on samples of biological and nonbiological origins containing ENMs (for example, metal and metal oxide nanoparticles, or carbon nanotubes, or both). This guide does not cover or address provisions for imaging or identifying, or both, non-engineered (natural) nanoparticles/nanomaterials in cells or other matrices, nor does this guide describe or discuss the application of DFM/HSI for determining the dimensions of ENMs.
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1.1 This guide has been prepared to familiarize laboratory scientists with the background information and technical content necessary to image and identify engineered nanomaterials (ENMs) in cells via darkfield microscopy/hyperspectral imaging (DFM/HSI) methodology.  
1.2 DFM/HSI is a hyphenated bioanalytical technique/tool that combines optical microscopy with high-resolution spectral imaging to both spatially localize the distribution of and identify ENMs within a suitably prepared test sample.  
1.2.1 In the context of mammalian cells, ENMs will have distinctive light-scattering properties in comparison to subcellular organelles and cell structural features, which can allow one to discriminate between the spectral profiles of ENMs and cellular components.  
1.2.2 The light-scattering properties of ENMs in other test samples, such as fixed tissues, plants, complex drug product formulations, filter media, and so forth, will also be different from the native matrix component scattering signals inherent to these other types of samples, thus allowing for ENM visualization and identification.  
1.3 This guide is applicable to the use of DFM/HSI for identifying ENMs in the matrices mentioned.  
1.4 This guide describes and discusses basic practices for setting up and using DFM/HSI instrumentation, sample imaging techniques, considerations for optics, image analysis, and the use of reference spectral libraries (RSLs). DFM/HSI is routinely used in industry, academia, and government as a research and development and quality control tool in diverse areas of nanotechnology.  
1.5 The values stated in SI units are to be regarded as the standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of Internati...

SIGNIFICANCE AND USE
5.1 Au nano-objects in various shapes (that is, rods, particles) are increasingly used for a wide variety of applications. Medical applications of AuNPs, such as targeted drug delivery, tumor detection, and treatment are becoming more common (5). AuNPs have unique optical properties related to their size and their surface can be readily functionalized. Though Au is recognized to be inert and biocompatible in its bulk form, the behavior of Au nano-objects in biological systems and the environment must be tested to ensure their inertness and safety (6). It is important to know whether prepared and stored suspensions of AuNPs contain Au in its bound state (commonly Au (0) and particle adsorbed species) or ionized state (commonly, Au (I) or Au (III)) to attribute the biological response to the appropriate species. Krug, et al., concluded that the significance of toxicity studies is considerably reduced in those cases where the material properties of the nanomaterial suspensions were not characterized prior to and during the study (7). Furthermore, the analyte mass fraction of particle bound species is used with knowledge of particle size to compute particle number concentration.
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1.1 This test method describes the use of inductively coupled plasma optical emission spectrometry (ICP-OES; also includes ICP-AES, where AES is atomic emission spectrometry) or inductively coupled plasma mass spectrometry (ICP-MS) for the determination of the mass fraction of particle bound gold (Au) in colloidal Au suspensions. Particle bound Au is defined as the mass of Au associated with the nanoparticle (NP) fraction and strongly adsorbed to the particle surface. Unbound Au is the fraction of Au in the native suspension not associated with the Au nanoparticle fraction that is, the dissolved Au existing in solution as a complex or free ion. The mass fraction of particle bound Au is determined by subtracting the mass fraction of unbound Au measured in acidified subsamples of the particle-free supernatant from the total Au mass fraction measured in acid-digested subsamples of the colloidal Au suspension. The particle-free supernatant is obtained after centrifugation of the colloidal Au suspension. This standard prescribes the use of an appropriate internal standard and calibration using either external standardization or single-point standard additions.  
1.2 Colloidal gold suspensions with AuNP diameters ranging from 1 nm to 100 nm can be determined with this method.  
1.3 The standard is not limited to particles with a uniform Au composition and may be applicable to a core-shell particle with a Au shell treatment.  
1.4 This standard is specific to Au. The method may be applicable to other elements measurable by ICP-OES or ICP-MS but is limited to nanoparticles that are not reactive in aqueous suspension.  
1.5 No detailed instructions for operating instrumentation are provided because of differences among various makes and models. Instead, the analyst shall follow the instructions provided by the manufacturer of their particular ICP-OES, ICP-MS or centrifuge instrument, especially with regard to optimization of the instrument settings.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurements are included in this standard.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 PCS is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region. This guide highlights this light scattering technique, generally applicable in the particle size range from the sub-nm region until the onset of sedimentation in the sample. The PCS technique is usually applied to slurries or suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from other size diameters isolated by other techniques and users of the PCS technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques. Notwithstanding the preceding sentence, the technique is widely applied in industry and academia as both a research and development tool and as a QC method for the characterization of submicron systems.
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1.1 This guide deals with the measurement of particle size distribution of suspended particles, which are solely or predominantly sub-100 nm, using the photon correlation (PCS) technique. It does not provide a complete measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that should be followed for good practice, along with potential pitfalls.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
3.1 This standard is intended to facilitate communication among members of the business, research, legal, government, and educational communities.  
3.2 Definitions:  
3.2.1 Terms and their related standard definitions in Section 4 are intended for use uniformly and consistently in all nanotechnology test methods, specifications, guides, and practices. The purpose of such use is to promote a clear understanding and interpretation of the standards in which they are used.  
3.2.2 Definitions of terms are written in the broadest sense possible, consistent with the intended meaning using the following guidance considerations.
3.2.2.1 Terminology E1992 and Practice E1964 concepts are considered, especially Sections 6.5, 7, and 8 of Practice E1964.
3.2.2.2 Terms and nomenclature are based on observed scientific phenomena and are descriptive, distinguishable, and have significant currency in the nanotechnology field as reflected in peer-reviewed articles and other objective sources. These terms and names should not disrupt accepted usage in other scientific and technological fields, and their preferred usage should follow accepted scientific syntax.
3.2.2.3 When incorporating a term or name from a related field, its underlying meaning is not redefined. Modifications are minimal and are done to elucidate scientific distinctions required by nanotechnology practitioners.
3.2.2.4 When conflicting or overlapping terms and names arise between scientific disciplines, precedence was given to the established term that has behind it a significant body of knowledge.
3.2.2.5 The definition of a term that can have different meanings in different technical fields, especially those fields beyond nanotechnology, is preceded by a limiting phrase, for example, “in nanotechnology.”  
3.3 Description of Terms:  
3.3.1 Descriptions of Terms are special purpose definitions intended to provide a precise understanding and interpretation of standards in which they are used.  
3.3...
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1.1 Nanotechnology is an emerging field; this standard defines the novel terminology developed for its broad multi- and interdisciplinary activities. As the needs of this area develop, this standard will evolve accordingly. Its content may be referenced or adopted, or both, in whole or in part, as demanded by the needs of the individual user.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide a basic educational structure for pattern generation in nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this standard may require a post-secondary two-year science or technology background to be understood sufficiently.  
5.3 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of pattern generation methods, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.4 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
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1.1 This guide provides a framework for a basic workforce education in pattern generation topics related to nanotechnology, to be taught at an undergraduate college level. The education should be broadbased, preparing an individual to work in one of many areas in nanotechnology research, development, or manufacturing. The individual so educated may be involved in nanoscale pattern definition.  
1.2 This guide may be used to develop or evaluate an education program for pattern generation topics in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this standard guide, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the methods and concepts needed for pattern generation in nanotechnology. It may not cover knowledge and skill objectives applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This practice establishes the basic structure for education in the characterization of nanoscale materials at the undergraduate college level. The approach taken is to classify specific characterization methods into two tiers, with a minimum number of methods to be selected from each tier and taught at an in-depth or introductory level. This offers the flexibility of tailoring to regional industry needs while still retaining a high degree of equivalency in educational depth and breadth across geographical boundaries.  
5.2 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of characterization methods, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.3 This practice is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
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1.1 This practice describes a procedure to provide the basic education of characterization methods for nanometer-scale materials, to be taught at an undergraduate college level. This education should be broad and include a suite of characterization methods to prepare an individual to work in various capacities within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This practice may be used to develop or evaluate an education program for characterization in the nanotechnology field. It provides listings of key methods that are relevant to such a program, with a minimum number of these methods to be taught as a requirement for such an education. This practice does not provide specific course material to be used in such a program.  
1.3 While no units of measurements are used in this practice, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the characterization methods for nanometer-scale materials, nor is it meant for use in certification processes. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide is to provide, at the undergraduate college level, a basic educational structure in the health and safety aspects of nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently. Depth on the topics should be sufficient to transfer between various applications of nanotechnology such as nanomaterial fabrication, nanomaterial characterization, nanolithography, and patterning.  
5.3 Nanoscale materials might present unique health and environmental hazards due to their unique properties. The hazards, if any, presented by nanomaterials can be very different from those presented by bulk/macroscopic materials.  
5.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to keep current on the technology, and have a base education that enables synthesis of emerging safety procedures and practices.  
5.5 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of the health and safety aspects associated with working in a manufacturing or research setting, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.6 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evo...
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1.1 This guide provides a framework for basic workforce education in health and safety topics related to nanotechnology, to be taught at an undergraduate college level. This education should be broad to prepare an individual to work safely within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This guide may be used to develop or evaluate an education program for health and safety issues in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 The immediate and long term hazards, if any, of many nanomaterials are unknown. This guide does not address concerns with consumer usage and eventual disposal of products that contain nanomaterials.  
1.5 This standard does not purport to address all of the techniques, materials, and concepts associated with health and safety topics related to nanotechnology. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally reco...

SIGNIFICANCE AND USE
5.1 Particle size is a key property of manufactured or engineered nanoparticles used in a wide range of applications. For purposes relevant to evaluations of safety, effectiveness, performance, quality, public health impact, or regulatory status of products, the correct measurement and uniform reporting of size and related parameters under use conditions, or during the manufacturing process, are critical to suppliers, analysts, regulators and other stakeholders.  
5.2 This test method is intended principally for the analysis of nanoparticles in aqueous suspension with dimensions between about 1 nm and 100 nm, but may be applied to diffusive colloidal particles even if their dimensions fall outside the nanoscale range (up to 1000 nm).  
5.3 For more detailed guidance on DLS measurements, including operational aspects, refer to Appendix X2 of this test method.
Note 1: The user is also referred to Guide E2490, which provides broad guidance for the application of DLS to nanomaterials. Guide E2490 is not required for the implementation of this test method.
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1.1 This test method addresses the determination of nanoparticle size (equivalent sphere hydrodynamic diameter) using batch-mode (off-line) dynamic light scattering (DLS) in aqueous suspensions and establishes general procedures that are applicable to many commercial DLS instruments. This test method specifies best practices, including sample preparation, performance verification, data analysis and interpretation, and reporting of results. The document includes additional general information for the analyst, such as recommended settings for specific media, potential interferences, and method limitations. Issues specific to the use of DLS data for regulatory submissions are addressed.  
1.2 The procedures and practices described in this test method, in principle, may be applied to any particles that exhibit Brownian motion and are kinetically stable during the course of a typical experimental time frame. In practice, this includes particles up to about 1000 nm in diameter, subject to limitations as described in the test method.  
1.3 This test method does not provide test specimen preparation procedures for all possible materials and applications, nor does it address synthesis or processing prior to sampling. The test specimen (suspension) preparation procedures should provide acceptable results for a wide range of materials and conditions. The analyst must validate the appropriateness for their particular application.  
1.4 This test method is applicable to DLS instruments that implement correlation spectroscopy. Analysts using instruments based on frequency analysis may still find useful information relevant to many aspects of the measurement process, including limits of applicability and best practices. On-line (flow-mode) DLS measurements are not treated here specifically and may have additional limitations or issues relative to batch-mode operation.  
1.5 Units—The values stated in SI units are to be regarded as standard. Where appropriate, c.g.s. units are given in addition to SI.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 The remarkable structural, physical and chemical properties of graphene — particularly its mechanical strength, high electronic mobility, lightness, and transparency (single layer or a few layers) — have generated worldwide research and industrial production efforts aimed at developing practical applications. Various industrially scalable production methods have been developed, including bottom-up approaches that grow graphene from small molecules (with or without a substrate), and top-down methods that start with graphite and exfoliate it by mechanical, chemical or electrochemical methods to produce nanoscale product such as graphene flakes. Two common exfoliation methods are: (1) oxidation of graphite to graphene oxide (GO) followed by additional processing to form reduced graphene oxide (r-GO) (2) and, (2) liquid phase exfoliation of graphite (3). The exfoliation methods, as well as substrate-less bottom-up approaches, produce materials in the form of flakes that can be dispersed in various solvents, making them suitable for applications requiring solution processing. Although there are many commercial “graphene” materials available on the market, the quality of these products is highly variable (4). There are many challenges in assessing the physical properties of the materials. In this guide we discuss how Raman spectroscopy (Raman) and X-ray photoelectron spectroscopy (XPS), as well as atomic force microscopy (AFM) can be used to characterize materials consisting of flakes of graphene and related materials (that is, few layer graphene (FLG), GO, r-GO). Illustrative examples are provided showing how these methods can be used to identify the type of material present and to extract important parameters including lateral flake size, average flake thickness, ratio of intensities of the D and G modes (ID/IG) in the Raman spectrum and carbon to oxygen ratio. Specifically, when encountering an “unknown” material or product purporting to be “graphene,” it is essent...
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1.1 This standard will provide guidance on the measurement approaches for assessment of lateral flake size, average flake thickness, Raman intensity ratio of the D to G bands, and carbon/oxygen ratio for graphene and related products. The techniques included here are atomic force microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy. Examples will be given for each type of measurement.  
1.2 This guide is intended to serve as an example for manufacturers, producers, analysts, and others with an interest in graphene and related products such as graphene oxide and reduced graphene oxide. This Standard Guide is not intended to be a comprehensive overview of all possible characterization methods.  
1.3 This guide does not include all sample preparation procedures for all possible materials and applications. The user must validate the appropriateness for their particular application.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This test method will assess whether the test nanoparticulate material has chemoattractant activity.  
5.2 This test method will provide a rapid and quantitative measure of the ability of nanoparticulate material to recruit immune cells.  
5.3 Recruitment of immune cells by chemotaxis plays an important part in all phases of both humoral and cell-mediated immune responses.  
5.4 Testing the capacity of a nanoparticulate material to recruit immune cells in vitro helps in predicting the influence of such material on the immune cell response.
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1.1 This test method provides a protocol for rapid and quantitative measurement of the chemoattractant capacity of a nanoparticulate material (nanoparticles and their aggregates and agglomerates).  
1.2 Immune cells recruitment (by chemotaxis) plays a central role in the immune system function especially in the inflammatory process.  
1.3 This test method uses an in vitro model. In this model, peripheral blood human acute promyelocytic leukemia cells HL-60 are separated from control chemoattractant or test nanoparticulate material by a 3-µm pore size filter; the cell migration through the filter is monitored and quantified using the fluorescent dye calcein AM (Figs. 1 and 2).
FIG. 1 Chemotaxis Chamber (Boyden Chamber)
FIG. 2 Chemotaxis Assay
a (left)—Parts of the chemotaxis assay assembly.
b (right)—Procedure for testing the chemoattractant capacity of a nanoparticulate material.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 A nano-object is an individual, well-defined, and separable piece of a nanomaterial; in practice, nanomaterials used in research, products, testing, and other uses are almost always collections of nano-objects. Individual nano-objects and collections of nano-objects are the two major types of nanomaterials in use. The description of a collection of nano-objects is covered in this guide. The description of an individual nano-object is covered in Guide E3144. Nano-objects, individually or as a collection, are often embedded in other materials for commercial, research, and other uses. These embedded nanomaterials can be described using the information categories and descriptors included in these guides.  
5.2 Nanomaterials are of growing importance in research and commerce, and data on their physical and chemical characteristics are critical to predict performance, to transact commercial activities, to assess their potential for harm to human and animal health and the environment in general, and to support regulations that affect their use. A collection of nano-objects is the predominant type of nanomaterial tested and used in commerce.  
5.3 The four types of data and information used to describe a collection of nano-objects are: physical and chemical characteristics; production; specifications; and general identifiers (names and classifications), as shown in Fig. 1.  
FIG. 1 Data and Information Types Necessary to Describe a Collection of Nano-Objects
Used with permission of CODATA from “Uniform Description System for Materials on the Nanoscale,” Version 2.0, http://doi.org/10.5281/zenodo.56720.  
5.4 This guide deals solely with data and information to describe the physical and chemical characterization of a collection of individual nano-objects.
Note 2: Other guides and documents (see Section 2, Referenced Documents) deal with the data and information for production, specifications, and general identifiers (names and classifications).
Note 3: Specifi...
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1.1 This guide provides guidelines for a description system to report the physical and chemical characteristics of collections of nano-objects. It establishes information categories and descriptors useful in describing collections of nano-objects uniquely and such that the equivalency of two or more collections of nano-objects can be determined according to specific criteria.  
1.2 This guide is designed to be directly applicable to reporting the physical and chemical characteristics of collections of nano-objects in most circumstances, including, but not limited to, reporting original research results in the archival literature, developing ontologies, database schemas, data repositories, and data reporting formats, specifying regulations, and enabling commercial activity.  
1.3 This guide is applicable to collections of naturally occurring, engineered, and manufactured nano-objects.  
1.4 One goal of the guide is to help ensure that when measurement results are reported, they are reported uniformly.  
1.5 A second goal of the guide is to encourage reports on the properties and functionalities of a collection of nano-objects to include as much detail as possible about the physical and chemical characteristics of that collection so it is uniquely specified.  
1.6 This guide does not cover the chemical reactions or reactivity of a collection of nano-objects.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization establishe...

SIGNIFICANCE AND USE
5.1 A nano-object is an individual, well-defined, and separable piece of a nanomaterial; in practice, nanomaterials used in research, products, testing, and other uses are usually collections of nano-objects. Individual nano-objects and collections of nano-objects are the two major types of nanomaterials in use. The description of an individual nano-object is covered in this guide; the description of collections of nano-objects (for example, two or more individual nano-objects) is not covered in this guide.  
5.2 Nanomaterials are of growing importance in research and commerce, and data on their physical and chemical characteristics are critical to predict performance, to transact commercial activities, to assess their potential for harm to human and animal health and the environment in general, and to support regulations that affect their use. To describe nanomaterials, whether for research or commerce, it is important to be able to describe an individual nano-object.  
5.3 When in a liquid environment, a nano-object may attract a non-permanent “halo” of water or other solvent molecules; such a non-permanent halo is not described by the present guide.  
5.4 In contrast, coatings and coronas are semi-permanent or permanent adherents on time scales commensurate with testing procedures; coatings and coronas can fundamentally transform one nano-object to another in terms of its characteristics and can be described using this guide.  
5.5 The four types of data and information used to describe a nano-object are: physical and chemical characteristics; production; specifications; and general identifiers (names and classifications), as shown in Fig. 1.  
5.6 This guide deals solely with data and information to describe the physical and chemical characterization of an individual nano-object.
Note 2: Other guides and documents (see Section 2, Referenced Documents) deal with the data and information for production, specifications, and general identifiers (names and cla...
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1.1 This guide provides guidelines for a description system for reporting the physical and chemical characteristics of individual nano-objects. It establishes information categories and descriptors useful in describing individual nano-objects uniquely and such that the equivalency of two or more individual nano-objects can be determined according to specific criteria.  
1.2 This guide is designed to be directly applicable to reporting the physical and chemical characteristics of nano-objects in every circumstance, including but not limited to reporting original research results in the archival literature, developing ontologies, database schemas, data repositories, and data reporting formats, specifying regulations, and enabling commercial activity.  
1.3 This guide is applicable to naturally-occurring, engineered, and manufactured nano-objects.  
1.4 One goal of the guide is to help ensure that when measurement results are reported, they are reported uniformly.  
1.5 A second goal of the guide is to encourage reports on the properties and functionalities of a nano-object to include as much detail as possible about the physical and chemical characteristics of that nano-object so it is uniquely specified.  
1.6 This guide does not cover the chemical reactions or reactivity of a nano-object.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International ...

SIGNIFICANCE AND USE
5.1 This guide is intended for use by entities involved in the handling of UNP in occupational settings. This guide covers handling principles and techniques that may be applied, as appropriate, to the variety of UNP materials and handling settings. These settings include research and development activities, material manufacturing, and material use and processing. This guide may also be used by entities that receive materials or articles containing or comprising nanoscale particles fixed upon or within a matrix (that is, bound nanoscale particles), but whose own processes or use may reasonably be expected to cause such particles to become unbound.
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1.1 This guide describes actions that could be taken by the user to minimize human exposures to unbound, engineered nanoscale particles (UNP) in research, manufacturing, laboratory and other occupational settings where UNP may reasonably be expected to be present. It is intended to provide guidance for controlling such exposures as a cautionary measure where neither relevant exposure standards nor definitive hazard and exposure information exist.  
1.2 General Guidance—This guide is applicable to occupational settings where UNP may reasonably be expected to be present. Operations across those settings will vary widely in the particular aspects relevant to nanoscale particle exposure control. UNP represent a vast variety of physical and chemical characteristics (for example, morphology, mass, dimension, chemical composition, settling velocities, surface area, surface chemistry) and circumstances of use. Given the range of physical and chemical characteristics presented by the various UNP, the diversity of occupational settings and the uneven empirical knowledge of and experience with handling UNP materials, the purpose of this guide is to offer general guidance on exposure minimization approaches for UNP based upon a consensus of viewpoints, but not to establish a standard practice nor to recommend a definite course of action to follow in all cases.  
1.2.1 Accordingly, not all aspects of this guide may be relevant or applicable to all circumstances of UNP handling. The user should apply reasonable judgment in applying this guide including consideration of the characteristics of the particular UNP involved, the user’s engineering and other experience with the material, and the particular occupational settings where the user may apply this guide. Users are encouraged to obtain the services of qualified professionals in applying this guide.  
1.2.2 Applicable Where Relevant Exposure Standards Do Not Exist—This guide assumes that the user is aware of and in compliance with any authoritative occupational exposure standard applicable to the bulk form of the UNP. This guide may be appropriate where such exposure standards do not exist, or where such standards exist, but were not developed with consideration of the nanoscale form of the material.  
1.3 Applicable Where Robust Risk Information Does Not Exist—This guide assumes the absence of scientifically sound risk assessment information relevant to the particular UNP involved. Where sound risk assessment information exists, or comes to exist, any exposure control measures should be designed based on that information, and not premised on this guide. Such measures may be more or less stringent than those suggested by this guide.  
1.4 Materials Within Scope—This guide pertains to unbound engineered nanoscale particles or their respirable agglomerates or aggregates thereof. Relevant nanoscale particle types include, for example, intentionally produced fullerenes, nanotubes, nanowires, nanoropes, nanoribbons, quantum dots, nanoscale metal oxides, and other engineered nanoscale particles. Respirable particles are those having an aerodynamic equivalent diameter (AED) less than or equal to 10 µm (10 000 nm) or those particles small enough to be collected with a respirable sampler (1-3).2 The AED desc...

SIGNIFICANCE AND USE
5.1 A nano-object at any specific time can be considered well-defined.  
5.2 The life-cycle of a nano-object can be viewed as a series of production processes that transforms starting materials or a well-defined nano-object into a new, equally well-defined nano-object.  
5.3 Each step of the life-cycle can be considered a separate production action and can be described by the information categories and descriptors within this guide.  
5.4 The following are examples of nano-object productions that can be described by this guide.  
5.4.1 The creation of carbon nanotubes by arc discharge.  
5.4.2 The coating of a nano-object in a random or controlled manner when placed in a liquid.
Note 1: The reactivity of nano-objects makes it likely that even with the utmost precautions, various features and characteristics may change over time, for example, when a nano-object is placed in a liquid and coated. Such a coating can significantly change the properties, functionalities, and reactivity of the nano-object. This change can be considered one step of a life-cycle and is a production process.
Note 2: A nano-object may have more than one coating. For example, titania nano-objects are often coated by alumina by manufacturers to control certain properties. When these previously coated nano-objects are placed in liquid containing biological molecules, they can acquire a second coating. It can require very careful administration of test procedures to ensure the test results can meaningfully be ascribed to characteristics and features of the “initial” nano-objects.  
5.4.3 A nano-object experiences changes to its size, shape, physical structure, and other characteristics.
Note 3: Events such as shock (unexpected forces), temperature and pressure changes, humidity changes, shipping, dissolution, and exposure to acids and bases can result in a changed nano-object with significantly different properties, functionalities, and reactivity. These events can be considered a prod...
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1.1 This guide provides guidelines for describing the production of one or more individual nano-objects. It establishes essential and desirable information categories and descriptors important to specify the production process, including the starting materials, the process itself, and the resulting nano-objects.  
1.2 This guide is designed to be directly applicable to reporting production information and data for nano-objects in most circumstances, including but not limited to reporting original research results in the archival literature, developing of ontologies, database schemas, data repositories and data reporting formats, specifying regulations, and enabling commercial activity.  
1.3 This guide is applicable to an individual nano-object and a collection of nano-objects.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Lipid composition in a liposomal formulation is an important aspect during synthesis of liposomes, which determines stability, surface characteristics, drug encapsulation, and drug release capabilities. The cholesterol component plays a key role in controlled drug release by adding stability to the liposome. A small variation in the lipid composition can significantly alter the parameters mentioned above (15).  
5.2 Variation in the lipid composition in the liposomal formulation may influence the safety and efficacy of the product. Therefore, chemical composition of the liposomes shall be determined.  
5.3 The pharmaceutical industry and regulatory agencies require QC, QA, specifications, thorough characterization, and quantification of lipid components (16, 17).  
5.4 This test method can be used to ascertain variations in the lipid component profiling of various liposomal formulations. However, this test method does not intend to identify chemical degradation products (18).  
5.5 Analyzing the stability of analytes and their chemical degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method (18, 19).
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1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal formulations as major components.  
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and evaporative light-scattering detection (ELSD) to quantify the individual components.  
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-phosphatidylcholine (HSPC).  
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.  
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method development and method validation, sample analysis, and data reporting.  
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 2 to 4 µg/g and 7 to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC are 10 to 165 µg/g, 10 to 300 µg/g, and 10 to 200 µg/g, respectively.  
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in Practice D6026.  
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.  
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Silver may be used to treat consumer textile products to provide enhanced antimicrobial (fungi, bacteria, viruses) properties (3, 4). At any point in a textile product’s lifecycle, there may be a need to measure the amount of silver present. This standard prescribes a test method based on ICP-OES or ICP-MS analysis that manufacturers, producers, analysts, policymakers, regulators, and others may use for measurement of total silver in textiles. As described in Guide E3025, determination of total silver in a consumer textile product is one component of a tiered approach to determine if silver is present, possibly as nanomaterial(s) (one or more external dimensions in the nanoscale), prior to measuring the form and dimension of the Ag that is found. ICP-OES or ICP-MS analysis alone is not sufficient to determine whether a textile contains silver nanomaterial(s).
Note 4: There are many different chemical and physical forms of silver that are used to treat textiles and an overview of this topic is provided in Guide E3025.  
5.2 As described in Guide E3025, the amount of silver in a textile can decrease over time as silver metal and silver compounds can react with oxygen and other oxidation-reduction (redox) active agents present in the environment to form soluble ionic species which are released by contact with moisture (for example, from ambient humidity, washing, body sweat, rain, or other sources). Hence, if silver is measured in a textile, the result may only be indicative of that moment in the article’s life cycle and great care is necessary in drawing temporal inferences from the results.  
5.3 If silver is measured by ICP-OES or ICP-MS analysis, additional analyses are needed to elucidate the form of silver in the textile specimen. This step is necessary because ICP-OES or ICP-MS results are for total silver independent of chemical and physical form and textiles may be treated with silver in sizes that range from the nanoscale (for example, salt nanopartic...
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1.1 This test method covers the use of inductively coupled plasma–optical emission spectrometry (ICP-OES) and inductively coupled plasma–mass spectrometry (ICP-MS) analyses for determination of the mass fraction of total silver in consumer textile products made of any combination of natural or manufactured fibers. Either ICP-OES or ICP-MS analysis is recommended as a first step to test for and quantify silver in a textile and results can be used to inform subsequent, more detailed analyses as part of the tiered approach described in Guide E3025 to determine if a textile contains silver nanomaterial(s).  
1.2 This test method prescribes acid digestion to prepare test sample solutions from samples of textiles utilizing an appropriate internal standard followed by external calibration and analysis with either ICP-OES or ICP-MS to quantify total silver.  
1.3 This test method is believed to provide quantitative results for textiles made of fibers of rayon, cotton, polyester, and lycra that contain metallic silver (see Section 17). It is the analyst’s responsibility to establish the efficacy (ability to achieve the planned and desired analytical result) of this test method for other textile matrices and forms of silver.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurements are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organiz...

SIGNIFICANCE AND USE
5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.
FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEM
Note 1: Both images are shown to the same scale; scale bar is 200 nm.  
5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same liposomal preparation is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.  
5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in a cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface o...
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1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 A tiered strategy for characterization of nanoparticle properties is necessary to draw meaningful conclusions concerning dose-response relationships observed during inhalation toxicology experiments. This tiered strategy includes characterization of nanoparticles as produced (that is, measured as the bulk material sold by the supplier) and as administered (that is, measured at the point of delivery to a test subject) (Oberdorster et al. (6)).  
5.2 Test Methods B922 and C1274 and ISO 9277 and ISO 18757 exist for determination of the as produced surface area of bulk metal and metal oxide powders. During the delivery of nanoparticles in inhalation exposure chambers, the material properties may undergo change and therefore have properties that differ from the material as produced. This test method describes the determination of the as administered surface area of airborne metal oxide nanoparticles in inhalation exposure chambers for inhalation toxicology studies.
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1.1 This test method covers determination of surface area of airborne metal oxide nanoparticles in inhalation exposure chambers for inhalation toxicology studies. Surface area may be measured by gas adsorption methods using adsorbates such as nitrogen, krypton, and argon (Brunauer et al. (1),2 Anderson (2), Gregg and Sing (3)) or by ion attachment and mobility-based methods (Ku and Maynard (4)). This test method is specific to the measurement of surface area by gas adsorption by krypton gas adsorption. The test method permits the use of any modern commercial krypton adsorption instruments but strictly defines the sample collection, outgassing, and analysis procedures for metal and metal oxide nanoparticles. Use of krypton is required due to the low overall surface area of particle-laden samples and the need to accurately measure the background surface area of the filter used for sample collection. Instrument-reported values of surface area based on the multipoint Brunauer, Emmett and Teller (BET) equation (Brunauer et al. (1), Anderson (2), Gregg and Sing (3)) are used to calculate surface area of airborne nanoparticles collected on a filter.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. State all numerical values in terms of SI units unless specific instrumentation software reports surface area using alternate units.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.
FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEM
Note 1: Both images are shown to the same scale; scale bar is 200 nm.  
5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same sample is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.  
5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface of the grid, and s...
SCOPE
1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Cryo-TEM is a technique used to record high resolution images of samples that are frozen and embedded in a thin layer of vitrified, amorphous ice (2-5). Because vitrification occurs so rapidly, the resultant specimen is almost instantly frozen, yielding a very accurate representation of the specimen at the moment of freezing, without the distortions typically associated with air drying delicate wet samples. Once frozen, images of the specimen are recorded at low temperature using a specially designed electron microscope equipped with a cryo-holder capable of operating under low dose conditions in order to prevent beam induced structural damage to the specimen. The cryo-TEM technique is the consensus choice to directly observe, analyze and accurately measure liposomes suspended in aqueous solutions. Fig. 1 illustrates this by comparing an electron micrograph from an air-dried negatively stained liposomal preparation with an electron micrograph of the same solution imaged by cryo-TEM.
FIG. 1 Left—An Electron Micrograph of an Air-Dried Liposomal Preparation that has been Negatively Stained with 2 % Uranyl Acetate for Contrast; Right—An Electron Micrograph of the Same Liposomal Preparation Prepared as a Frozen Vitrified Specimen for Cryo-TEM
Note 1: Both images are shown to the same scale; scale bar is 200 nm.  
5.1.1 Fig. 1 demonstrates that liposomes may become distorted and are difficult to measure and analyze when they are air-dried, while the same sample is clearly easier to analyze when the specimen is near-instantly preserved by vitrification.  
5.1.2 Cryo-TEM involves applying a small volume of sample to a specially prepared holey, ultra-thin or continuous carbon grid suspended in cryo-TEM plunger over a cup of liquid ethane cooled in a container filled with liquid nitrogen (2, 3). These grids can be purchased or prepared in the laboratory using a carbon evaporator with glow discharge capabilities. Once the sample has wet the surface of the grid, and s...
SCOPE
1.1 This practice covers procedures for vitrifying and recording images of a suspension of liposomes with a cryo-transmission electron microscope (cryo-TEM) for the purpose of evaluating their shape, size distribution and lamellarity for quality assessment. The sample is vitrified in liquid ethane onto specially prepared holey, ultra-thin, or continuous carbon TEM grids, and imaged in a cryo-holder placed in a cryo-TEM.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 Mean particle diameters defined according to the Moment-Ratio (M-R) system are derived from ratios between two moments of a particle size distribution.
SCOPE
1.1 The purpose of this practice is to present procedures for calculating mean sizes and standard deviations of size distributions given as histogram data (see Practice E1617). The particle size is assumed to be the diameter of an equivalent sphere, for example, equivalent (area/surface/volume/perimeter) diameter.  
1.2 The mean sizes/diameters are defined according to the Moment-Ratio (M-R) definition system.2,3,4  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SCOPE
1.1 This guide deals with the measurement of mobility and zeta potential in systems containing biological material such as proteins, DNA, liposomes and other similar organic materials that possess particle sizes in the nanometer scale (  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 NTA is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region. This guide describes the NTA technique for direct visualization and measurement of Brownian motion, generally applicable in the particle size range from several nanometers until the onset of sedimentation in the sample. The NTA technique is usually applied to dilute suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from size diameters determined by other techniques and users of the NTA technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques (see 8.7). Notwithstanding the preceding sentence, the technique is routinely applied in industry and academia as both a research and development tool and as a QC method for the characterization of submicron systems.
SCOPE
1.1 This guide deals with the measurement of particle size distribution of suspended particles, from ~10 nm to the onset of sedimentation, sample dependent, using the nanoparticle tracking analysis (NTA) technique. It does not provide a complete measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that should be followed for good practice, along with potential pitfalls.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide establishes, at the undergraduate college level, the basic education structure for understanding the unique properties and applications of nanoscale materials as compared to bulk properties and applications of macroscale materials.  
5.2 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of material properties and the effects of size, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.3 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
SCOPE
1.1 This guide provides a framework for a basic workforce education in material properties at the nanoscale, to be taught at an undergraduate college level. This education should be broad to prepare an individual to serve within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This guide may be used to develop or evaluate an education program for unique material properties and their applications in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this guide, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This standard does not purport to address all of the techniques, materials, and concepts needed for material properties and applications. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 As AFM measurement technology has matured and proliferated, the technique has been widely adopted by the nanotechnology research and development community to the extent that it is now considered an indispensible tool for visualizing and quantifying structures on the nanoscale. Whether used as a stand-alone method or to complement other dimensional measurement methods, AFM is now a firmly established component of the nanoparticle measurement tool box. International standards for AFM-based determination of nanoparticle size are nonexistent as of the drafting of this guide. Therefore, this standard aims to provide practical and metrological guidance for the application of AFM to measure the size of substrate-supported nanoparticles based on maximum displacement as the probe is rastered across the particle surface to create a line profile.
SCOPE
1.1 The purpose of this document is to provide guidance on the quantitative application of atomic force microscopy (AFM) to determine the size of nanoparticles2 deposited in dry form on flat substrates using height (z-displacement) measurement. Unlike electron microscopy, which provides a two-dimensional projection or a two-dimensional image of a sample, AFM provides a three-dimensional surface profile. While the lateral dimensions are influenced by the shape of the probe, displacement measurements can provide the height of nanoparticles with a high degree of accuracy and precision. If the particles are assumed to be spherical, the height measurement corresponds to the diameter of the particle. In this guide, procedures are described for dispersing gold nanoparticles on various surfaces such that they are suitable for imaging and height measurement via intermittent contact mode AFM. Generic procedures for AFM calibration and operation to make such measurements are then discussed. Finally, procedures for data analysis and reporting are addressed. The nanoparticles used to exemplify these procedures are National Institute of Standards and Technology (NIST) reference materials containing citrate-stabilized negatively charged gold nanoparticles in an aqueous solution.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide establishes the basic structure for education in the synthesis and processing of nanoscale materials at the undergraduate college level.  
5.2 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of synthesis and processing topics, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.3 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
SCOPE
1.1 This guide provides a framework for a basic workforce education in materials synthesis and processing at the nanoscale, to be taught at an undergraduate college level. This education should be broad to prepare an individual to serve within one of the many areas in nanotechnology research, development, or manufacturing.  
1.2 This guide may be used to develop or evaluate an education program for synthesis and processing applications in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this practice, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.5 This standard does not purport to address all of the techniques, materials, and concepts needed for materials synthesis and processing at the nanoscale. It is the responsibility of the user of this standard to utilize other knowledge and skill objectives as applicable to local conditions or required by local regulations.

SIGNIFICANCE AND USE
4.1 Natural and manufactured textiles fibers can be treated with chemicals to provide enhanced antimicrobial (fungi, bacteria, viruses) properties. In some cases, silver nanomaterials may be used to treat textile fibers (1).6 Silver nanomaterials are used to treat a wide array of consumer textile products, including but not limited to various clothing; primary garments (shirts, pants), outer wear (gloves, jackets), inner wear (socks and underwear), children’s clothing (sleepwear); children’s plush toys; bath towels and bedding (sheets, pillows); and medical devices (wound dressings) (2).  
4.2 There are many different chemical and physical forms of silver that are used to treat textiles and an overview of this topic is provided in Appendix X1.  
4.3 Several applicable techniques for detection and characterization of silver are listed and described in Appendix X2 so that users of this guide may understand the suitability of a particular technique for their specific textile and silver measurement need.  
4.4 There are many different reasons to assay for silver nanomaterials in a textile at any point in a product’s life cycle. For example, a producer may want to verify that a textile meets their internal quality control specifications or a regulator may want to understand the properties of silver nanomaterials used to make a consumer textile product under their jurisdiction or what quantity of silver nanomaterial is potentially available for release from the treated textile during a washing process. Regardless of the specific reason, a structured approach to detect and characterize silver nanomaterials present in a textile will facilitate measurements and data comparison.  
4.5 The approach presented in this guide (see Fig. 1) consists of three sequential tiers: obtain a textile sample (Section 7), detection of a silver nanomaterial (Section 8), and characterization of a silver nanomaterial (Section 9). If no forms of silver are detected in a textile sample using a...
SCOPE
1.1 This guide covers the use of a tiered approach for detection and characterization of silver nanomaterials in consumer textile products made of any combination of natural or manufactured fibers.  
1.2 This guide covers, but is not limited to, fabrics and parts (for example, thread, batting) used during the manufacture of textiles and production of consumer textile products that may contain silver-based nanomaterials. It does not apply to analysis of silver nanomaterials in non-consumer textile product matrices nor does it cover thin film silver coatings with only one dimension in the nanoscale.  
1.3 This guide is intended to serve as a resource for manufacturers, producers, analysts, policymakers, regulators, and others with an interest in textiles.  
1.4 This guide is presented in the specific context of measurement of silver nanomaterials; however, the structured approach described herein is applicable to other nanomaterials used to treat consumer textile products.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide a basic educational structure in the infrastructure aspects of nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing, nanomaterials characterization, or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently. Depth on the topics should be sufficient to transfer between various applications of nanotechnology such as nanomaterial fabrication, nanomaterial characterization, nanolithography, and patterning.  
5.3 Nanoscale materials might present unique health and environmental hazards due to their unique properties. The hazards, if any, presented by nanomaterials can be very different from those presented by bulk/macroscopic materials.  
5.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to keep current on the technology and have a base education that enables synthesis of emerging safety procedures and practices.
SCOPE
1.1 This document provides guidelines for basic workforce education in the infrastructure topics related to nanotechnology to be taught at an undergraduate college level. This education should be broad to prepare an individual to work within one of the many areas in nanotechnology research, development, or manufacturing. The individual so educated may be involved in material handling, manufacture, distribution, storage, use, or disposal of nanoscale materials.  
1.2 This guide may be used to develop or evaluate an education program for the infrastructure used in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.5 This standard does not purport to address all of the methods and concepts pertaining to the infrastructure for nanotechnology. It may not cover knowledge and skill objectives applicable to local conditions or required by local regulations.

SIGNIFICANCE AND USE
5.1 The purpose of this guide is to provide a basic educational structure for pattern generation in nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanufacturing or nanomaterials research.  
5.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this standard may require a post-secondary two-year science or technology background to be understood sufficiently. Depth on the topics should be sufficient to transfer between various applications of nanotechnology such as nanomaterial fabrication, nanomaterial characterization, nanolithography, and patterning.  
5.3 Workers may transition in their roles in the workplace. Participants in such education will have a broad understanding of a complement of pattern generation methods, thus increasing their marketability for jobs within as well as beyond the nanotechnology field.  
5.4 This guide is intended to be one in a series of standards developed for workforce education in various aspects of nanotechnology. It will assist in providing an organization a basic structure for developing a program applicable to many areas in nanotechnology, thus providing dynamic and evolving workforce education.
SCOPE
1.1 This guide provides a framework for a basic workforce education in pattern generation topics related to nanotechnology, to be taught at an undergraduate college level. The education should be broadbased, preparing an individual to work in one of many areas in naotechnology research, development, or manufacturing. The individual so educated may be involved in nanoscale pattern definition.  
1.2 This guide may be used to develop or evaluate an education program for pattern generation topics in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 While no units of measurements are used in this standard guide, values stated in SI units are to be regarded as standard.  
1.4 This standard does not purport to address all of the methods and concepts needed for pattern generation in nanotechnology. It may not cover knowledge and skill objectives applicable to local conditions or required by local regulations.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 PCS is one of the very few techniques that are able to deal with the measurement of particle size distribution in the nano-size region. This Guide highlights this light scattering technique, generally applicable in the particle size range from the sub-nm region until the onset of sedimentation in the sample. The PCS technique is usually applied to slurries or suspensions of solid material in a liquid carrier. It is a first principles method (that is, calibration in the standard understanding of this word, is not involved). The measurement is hydrodynamically based and therefore provides size information in the suspending medium (typically water). Thus the hydrodynamic diameter will almost certainly differ from other size diameters isolated by other techniques and users of the PCS technique need to be aware of the distinction of the various descriptors of particle diameter before making comparisons between techniques. Notwithstanding the preceding sentence, the technique is widely applied in industry and academia as both a research and development tool and as a QC method for the characterization of submicron systems.
SCOPE
1.1 This guide deals with the measurement of particle size distribution of suspended particles, which are solely or predominantly sub-100 nm, using the photon correlation (PCS) technique. It does not provide a complete measurement methodology for any specific nanomaterial, but provides a general overview and guide as to the methodology that should be followed for good practice, along with potential pitfalls.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 The purpose of this guide is to provide a basic educational structure in the health and safety aspects of nanotechnology to organizations developing or carrying out education programs for the nanotechnology workforce. This guide helps to describe the minimum knowledge base for anyone involved in nanomanfacturing or nanomaterials research.  
4.2 The basic education should prepare an individual for varied roles in the nanotechnology workplace. The material in this guide may require a post-secondary two-year science or technology background to be understood sufficiently. Depth on the topics should be sufficient to transfer between various applications of nanotechnology such as nanomaterial fabrication, nanomaterial characterization, nanolithography and patterning.  
4.3 Nanoscale materials might present unique health and environmental hazards due to their unique properties. The hazards, if any, presented by nanomaterials can be very different from those presented by bulk/macroscopic materials.  
4.4 Because nanotechnology is a rapidly developing field, the individual educated in nanotechnology needs to be cognizant of changing and evolving safety procedures and practices. Individuals should be aware of how to keep current on the technology, and have a base education that enables synthesis of emerging safety procedures and practices.
SCOPE
1.1 This guide provides guidelines for basic workforce education in health and safety topics related to nanotechnology, to be taught at an undergraduate college level. This education should be broad to prepare an individual to work safely within one of the many areas in nanotechnology research, development, or manufacturing. The individual so educated may be involved in material handling, manufacture, distribution, storage, use, or disposal of nanoscale materials.  
1.2 This guide may be used to develop or evaluate an education program for health and safety issues in the nanotechnology field. This guide provides listings of key topics that should be covered in a nanotechnology education program on this subject, but it does not provide specific course material to be used in such a program. This approach is taken in order to allow workforce education entities to ensure their programs cover the required material while also enabling these institutions to tailor their programs to meet the needs of their local employers.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. The immediate and long term hazards, if any, of many nanomaterials are unknown. This guide does not address concerns with consumer usage and eventual disposal of products that contain nanomaterials.  
1.5 This guide may not cover knowledge and skill objectives applicable to local conditions or required by local regulations.