E56.03 - Environment, Health, and Safety

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.
SCOPE
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.

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.
SCOPE
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 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.
SCOPE
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 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.
SCOPE
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 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.
SCOPE
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 des...

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.
SCOPE
1.1 This test method provides a protocol for quantitative analysis of the effect of nanoparticulate materials in physiologic solution on granulocyte-macrophage colony-forming units.  
1.2 This test method employs murine bone marrow hematopoietic stem cells which proliferate and differentiate to form discrete cell clusters or colonies which are counted.  
1.3 This test method is part of the in vitro preclinical characterization cascade for nanoparticulate materials for systemic administration in medical applications.  
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 and health practices and determine the applicability of regulatory limitations prior to use.

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 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.
SCOPE
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 represents potential target organs following systemic administration  
1.2 This test method is part of the 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 and health practices and determine the applicability of regulatory limitations prior to use.

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.
SCOPE
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 and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
Stem cells of hematopoietic origin are pluripotential and may be particularly sensitive to the effects of stimulation by nanoparticulate materials.
The effect of particles on macrophage responses has an extensive history and can be assessed by Practice F 1903. The test method described here will assess the effect on stem cells which can be progenitor cells to the macrophage line.
SCOPE
1.1 This test method provides a protocol for quantitative analysis of the effect of nanoparticulate materials in physiologic solution on granulocyte-macrophage colony-forming units.
1.2 This test method employs murine bone marrow hematopoietic stem cells which proliferate and differentiate to form discrete cell clusters or colonies which are counted.
1.3 This test method is part of the in vitro preclinical characterization cascade for nanoparticulate materials for systemic administration in medical applications.
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.

SIGNIFICANCE AND USE
Assessing the propensity of a nanomaterial to cause cytotoxicity to the cells of a target organ can assist in preclinical development.
The standard historical cytotoxicity testing of materials and extracts of materials has used fibroblasts and is well documented in Practice F 813, Test Method F 895, and ISO 10993-5. The use of macrophages and micron size particles has also provided information on cytotoxicity and stimulation using Practice F 1903.
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.
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 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.
SCOPE
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 represents potential target organs following systemic administration
1.2 This test method is part of the 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 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
p>This test method is one of a series of tests listed in Practice F 748 and ISO 10993-4 to assess the biocompatibility of materials contacting blood in medical applications.
This test method is similar to Practice F 756 but modified to accommodate nanoparticulate materials.
SCOPE
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 F 756 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 given in SI units are to be considered as the 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
This Guide is intended for use by entities involved in the handling of UNP in occupational settings. The 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. The Guide may also be used by entities that receive materials or articles containing or comprising nanoscale particles fixed upon or within a matrix (i.e., bound nanoscale particles), but whose own processes or use may reasonably be expected to cause such particles to become unbound.
SCOPE
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 (e.g., 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 users 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 m (10 000 nm) or those particles small enough to be collected with a respirable sampler (12, 34, 36). The AED describes the beha...