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ASTM E3251-20

(Test Method)

Standard Test Method for Microbial Ingress Testing on Single-Use Systems

Standard Test Method for Microbial Ingress Testing on Single-Use Systems

SIGNIFICANCE AND USE
4.1 Single-use systems (SUSs) used for biopharmaceutical manufacturing must maintain sterility and product quality of the fluid inside. Such articles or systems should therefore be validated as providing an effective barrier against microbial ingress. The microbial barrier properties of a SUS may be demonstrated using deterministic physical tests that have been correlated to microbial integrity. Two test methods (aerosol exposure and immersion exposure) are described that can be used to demonstrate microbial integrity of a SUS or determine the MALL, the maximum defect size that does not allow microbial ingress, into a SUS.  
4.2 It is important to note that the results of microbial ingress tests are heavily dependent on the conditions under which the test is performed and are not suitable for routine checking of a SUS due to the test’s destructive nature.  
4.2.1 Any size defect may be forced to fail under sufficiently aggressive conditions (including a large enough sample size, high differential pressure, or high hydrostatic pressure, for example) that would not ordinarily reflect normal use conditions. Thus, it is necessary to clearly define the relevant conditions for a test through a risk assessment of both the actual SUS claims and its final use (Practice E3244). Once that is established, the size of defect that can be detected under those conditions can be determined, if required, using defined defects.  
4.2.2 “Relevant conditions” refers to worse-case actual use conditions but does not mean that a SUS must be tested under theoretically absolute (extreme) “worst-case” conditions.  
4.2.3 Testing may be performed on individual components or entire systems. Considerations for defining “relevant conditions” and testing design should be based on a risk assessment for the SUS intended use and should include:
4.2.3.1 A channel created by a defect or breach through the film thickness or through a seam or connection which must be filled with liquid to allow mic...
SCOPE
1.1 The microbial test method outlined in this document applies to microbial ingress risk assessment of a single-use system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the assembly based on a potential risk of a breach to the product or manufacturing process.  
1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold:  
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use.  
1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing methods can be correlated to this MALL in order to claim microbial integrity. Test articles bearing calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a positive control (defect-based positive control), may be tested to determine the MALL.  
1.3 Both purposes for microbial ingress testing as described in 1.2.1 and 1.2.2 can either be conducted by liquid immersion or aerosol exposure. For the purpose described in 1.2.2, the type of exposure should be determined according to the SUS’s use-case conditions and a risk assessment.  
1.4 The method used to create a breach, hole or defect in single-use film or in a SUS test article, as ...

General Information

Status
Historical
Publication Date
30-Apr-2020
ICS
07.100.10 - Medical microbiology
Technical Committee
E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
Drafting Committee
E55.04 - General Biopharmaceutical Standards
Current Stage
Ref Project

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ASTM E3251-20 - Standard Test Method for Microbial Ingress Testing on Single-Use Systems
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E3251 − 20
Standard Test Method for
1
Microbial Ingress Testing on Single-Use Systems
This standard is issued under the fixed designation E3251; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope given test article should be justified with the rationale of
sampling size to obtain a statistically meaningful effect (Prac-
1.1 The microbial test method outlined in this document
tice E3244). Determining the appropriate number of SUS test
applies to microbial ingress risk assessment of a single-use
articles will depend on a risk assessment of the SUS and the
system (SUS) or its individual components that require integ-
conditions of its use and is also outside of this document’s
rity testing either by the assembly supplier or the end user of
scope.
the assembly based on a potential risk of a breach to the
product or manufacturing process. 1.5 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
1.2 The aim of microbial ingress testing of sterile SUSs
standard.
used in biopharmaceutical manufacturing is two-fold:
1.6 This standard does not purport to address all of the
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid
safety concerns, if any, associated with its use. It is the
path to remain sterile after a SUS has been challenged by
responsibility of the user of this standard to establish appro-
microbial exposure. Microbial exposure is achieved either by
priate safety, health, and environmental practices and deter-
directly placing a SUS into a container of microbial challenge
mine the applicability of regulatory limitations prior to use.
solution, or by delivering an aerosolized microbial challenge
1.7 This international standard was developed in accor-
onto a SUS that is placed inside a test chamber designed to
dance with internationally recognized principles on standard-
generate and deliver the aerosol. The choice of the test
ization established in the Decision on Principles for the
challenge organism should be justified based on a risk assess-
Development of International Standards, Guides and Recom-
ment of the SUS and conditions of use.
mendations issued by the World Trade Organization Technical
1.2.2 Additionally, microbial ingress testing can be used to
Barriers to Trade (TBT) Committee.
determine the maximum allowable leakage limit (MALL) that
does not allow microbial ingress under specific test conditions.
2. Referenced Documents
The defect size that can be detected by specific physical
2
2.1 ASTM Standards:
integrity testing methods can be correlated to this MALL in
order to claim microbial integrity. Test articles bearing cali- E3244 Practice for Integrity Assurance and Testing of
Single-Use Systems
brated defects over a range of dimensions, including up to a
defect size expected to consistently allow microbial ingress as 2.2 Other Documents:
USP <1207> Sterile Product Packaging — Integrity
a positive control (defect-based positive control), may be tested
3
to determine the MALL. Evaluation, 2016
4
ISO 15747 Plastic Containers for Intravenous Injections
1.3 Both purposes for microbial ingress testing as described
in 1.2.1 and 1.2.2 can either be conducted by liquid immersion
3. Terminology
or aerosol exposure. For the purpose described in 1.2.2, the
3.1 Definitions:
type of exposure should be determined according to the SUS’s
3.1.1 (calibrated) artificial defect, n—an artificial breach or
use-case conditions and a risk assessment.
defect (that is, laser-drilled hole, capillary) representing typical
1.4 The method used to create a breach, hole or defect in
failure modes, intentionally introduced into a test article.
single-use film or in a SUS test article, as well as the analytical
method used to physically characterize the defect size is
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
outside of the scope of this document. The sampling plan for a
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This test method is under the jurisdiction of ASTM Committee E55 on Available from U.S. Pharmacopeial Convention (USP), 12601 Twinbrook
Manufacture of Pharmaceutical and Biopharmaceutical Products and is the direct Pkwy., Rockville, MD 20852-1790, http://www.usp.org.
4
responsibility of Subcommittee E55.04 on General Biopharmaceutical Standards. Available from International Organization for Standardization (ISO), ISO
Current edition approved May 1, 2020.
...

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Standard Test Method for Microbial Ingress Testing on Single-Use Systems

SIGNIFICANCE AND USE
4.1 Single-use systems (SUSs) used for biopharmaceutical manufacturing must maintain sterility and product quality of the fluid inside. Such articles or systems should therefore be validated as providing an effective barrier against microbial ingress. The microbial barrier properties of a SUS may be demonstrated using deterministic physical tests that have been correlated to microbial integrity. Such physical test methods are described in Test Method E3336. Two microbial test methods (aerosol exposure and immersion exposure) are described in this test method that can be used to demonstrate microbial integrity of a SUS or determine the MALL, the maximum defect size that does not allow microbial ingress, into a SUS.  
4.2 It is important to note that the results of microbial ingress tests are heavily dependent on the conditions under which the test is performed and are not suitable for routine checking of a SUS due to the test’s destructive nature.  
4.2.1 Any size defect may be forced to fail under sufficiently aggressive conditions (including a large enough sample size, high differential pressure, or high hydrostatic pressure, for example) that would not ordinarily reflect normal use conditions. Thus, it is necessary to clearly define the relevant conditions for a test through a risk assessment of both the actual SUS claims and its final use (Practice E3244). Once that is established, the size of defect that can be detected under those conditions can be determined, if required, using defined defects.  
4.2.2 “Relevant conditions” refers to worse-case actual use conditions but does not mean that a SUS must be tested under theoretically absolute (extreme) “worst-case” conditions.  
4.2.3 Testing may be performed on individual components or entire systems. Considerations for defining “relevant conditions” and testing design should be based on a risk assessment for the SUS intended use and should include:
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SCOPE
1.1 The microbial test method outlined in this test method applies to microbial ingress risk assessment of a single-use system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the assembly based on a potential risk of a breach to the product or manufacturing process.  
1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold:  
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use.  
1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing methods (see Test Method E3336) can be correlated to this MALL in order to claim microbial integrity. Test articles bearing calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a positive control (defect-based positive control), may be tested to determine the MALL.  
1.3 Both purposes for microbial ingress testing as described in 1.2.1 and 1.2.2 can either be conducted by liquid immersion or aerosol exposure. For the purpose described in 1.2.2, the type of exposure should be determined according to the SUS’s use-case conditions and a risk assessment.  
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ASTM E3251-23(Test Method)
Standard Test Method for Microbial Ingress Testing on Single-Use Systems

SIGNIFICANCE AND USE
4.1 Single-use systems (SUSs) used for biopharmaceutical manufacturing must maintain sterility and product quality of the fluid inside. Such articles or systems should therefore be validated as providing an effective barrier against microbial ingress. The microbial barrier properties of a SUS may be demonstrated using deterministic physical tests that have been correlated to microbial integrity. Such physical test methods are described in Test Method E3336. Two microbial test methods (aerosol exposure and immersion exposure) are described in this test method that can be used to demonstrate microbial integrity of a SUS or determine the MALL, the maximum defect size that does not allow microbial ingress, into a SUS.  
4.2 It is important to note that the results of microbial ingress tests are heavily dependent on the conditions under which the test is performed and are not suitable for routine checking of a SUS due to the test’s destructive nature.  
4.2.1 Any size defect may be forced to fail under sufficiently aggressive conditions (including a large enough sample size, high differential pressure, or high hydrostatic pressure, for example) that would not ordinarily reflect normal use conditions. Thus, it is necessary to clearly define the relevant conditions for a test through a risk assessment of both the actual SUS claims and its final use (Practice E3244). Once that is established, the size of defect that can be detected under those conditions can be determined, if required, using defined defects.  
4.2.2 “Relevant conditions” refers to worse-case actual use conditions but does not mean that a SUS must be tested under theoretically absolute (extreme) “worst-case” conditions.  
4.2.3 Testing may be performed on individual components or entire systems. Considerations for defining “relevant conditions” and testing design should be based on a risk assessment for the SUS intended use and should include:
4.2.3.1 A channel created by a defect or breach through the...
SCOPE
1.1 The microbial test method outlined in this test method applies to microbial ingress risk assessment of a single-use system (SUS) or its individual components that require integrity testing either by the assembly supplier or the end user of the assembly based on a potential risk of a breach to the product or manufacturing process.  
1.2 The aim of microbial ingress testing of sterile SUSs used in biopharmaceutical manufacturing is two-fold:  
1.2.1 Firstly, it is used to evaluate the ability of a SUS fluid path to remain sterile after a SUS has been challenged by microbial exposure. Microbial exposure is achieved either by directly placing a SUS into a container of microbial challenge solution, or by delivering an aerosolized microbial challenge onto a SUS that is placed inside a test chamber designed to generate and deliver the aerosol. The choice of the test challenge organism should be justified based on a risk assessment of the SUS and conditions of use.  
1.2.2 Additionally, microbial ingress testing can be used to determine the maximum allowable leakage limit (MALL) that does not allow microbial ingress under specific test conditions. The defect size that can be detected by specific physical integrity testing methods (see Test Method E3336) can be correlated to this MALL in order to claim microbial integrity. Test articles bearing calibrated defects over a range of dimensions, including up to a defect size expected to consistently allow microbial ingress as a positive control (defect-based positive control), may be tested to determine the MALL.  
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Standard Test Method for Evaluation of Cytotoxicity of Nanoparticulate Materials in Porcine Kidney Cells and Human Hepatocarcinoma Cells

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.  
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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.  
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Standard Test Method for Evaluation of the Effect of Nanoparticulate Materials on the Formation of Mouse Granulocyte-Macrophage Colonies

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Standard Practice for Quantification of Calcium Deposits in Osteogenic Culture of Progenitor Cells Using Fluorescent Image Analysis

SIGNIFICANCE AND USE
5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcium deposits or mineralized matrix that form in vitro may be an indicator of differentiation to a functional osteoblast; however, expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast.  
5.2 This practice provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, XO. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to ensure that the images have not been underexposed or overexposed. Intensity and area do not correlate directly to calcium content.  
5.3 XO enables the monitoring of calcium deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of the mineralized area due to dye accumulation from repeated application (1).3 Calcium deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose (1) but require a different procedure for analysis than XO (that is, concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects.  
5.4 The practice may be applied to cultures of any cells capable of producing calcium deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization.  
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SCOPE
1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium-chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice.  
1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (that is, osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice.  
1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage (area%), for example, fraction of coverage of the area analyzed.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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ASTM E1326-20(Guide)
Standard Guide for Evaluating Non-culture Microbiological Tests

SIGNIFICANCE AND USE
5.1 This guide should be used by producers and potential producers of non-culture tests to determine the accuracy, selectivity, specificity, and precision of the tests, as defined in Practice E691. Results of such studies should identify the limitations and indicate the utility or applicability of the non-culture test, or both, for use on different types of samples. Guide E1488 recommends other statistical tools for evaluating the suitability and applicability of proposed new test methods.  
5.2 Non-culture test users and potential users should employ this guide to evaluate results of the non-culture test as compared to their present methods. Practices D5245 and D5465 should be reviewed in regards to the microbiological methods employed. If culture methods have not been used for monitoring the systems, then guidelines are included for obtaining microbiological expertise.  
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5.4 Detecting microbial contamination levels that exceed predetermined upper control limits indicates the need for an addition of an antimicrobial agent or other corrective maintenance action. By accurately determining this in a shorter time period than is possible than by culture methods, treatment with antimicrobial agents may circumvent more serious problems than if the treatment were postponed until culture results were available. If the antimicrobial treatment program relied on an inaccurate non-culture test, then unnecessary loss of product and problems associated with inappropriate selection or improper dosing with antimicrobial agents would exist.  
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SCOPE
1.1 The purpose of this guide is to assist users and producers of non-culture microbiological tests in determining the applicability of the test for processing different types of samples and evaluating the accuracy of the results. Culture test procedures such as the Heterotrophic (Standard) Plate Count, the Most Probable Number (MPN) method and the Spread Plate Count are widely cited and accepted for the enumeration of microorganisms. However, these methods have their limitations, such as performance time. Moreover, any given culture test method typically recovers only a portion of the total viable microbes present in a sample. It is these limitations that have recently led to the marketing of a variety of non-culture procedures, test kits and instruments.  
1.2 Culture test methods estimate microbial population densities based on the ability of mircoorganisms in a sample to proliferate in or on a specified growth medium, under specified growth conditions. Non-culture test methods attempt to provide the same or complimentary information through the measurement of a different parameter. This guide is designed to assist investigators in assessing the accuracy and precision of non-culture methods intended for the determination of microbial population densities or activities.  
1.3 It is recognized that the Heterotrophic Plate Count (HPC) does not recover all microorganisms present in a product or a system (1, 2).2 When this problem occurs during the characterization of a microbiological population, alternative standard enumeration procedures may be necessary, as in the case of sulfate-reducing bacteria. At other times, chemical methods that measure the rates of appearance of metabolic derivatives, the utilization of contaminated product components or genetic profile of the microbial population might be indicated. In evaluating non-culture tests, it is possible that the use of these alternative standard procedures might be...

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ASTM E1881-12(2020)(Guide)
Standard Guide for Cell Culture Analysis with SIMS

SIGNIFICANCE AND USE
5.1 The presence of cell growth medium complicates a direct analysis of cells with SIMS. Attempts to wash out the nutrient medium results in the exposure of cells to unphysiological reagents that may also alter their chemical composition. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided a unique way of sampling individual cells in their native state for SIMS analysis.  
5.2 The procedure described here has been successfully used for imaging Na+ and K+ ion transport  (3), calcium alterations in stimulated cells (4,5), and localization of therapeutic drugs and isotopically labeled molecules in single cells (6). The frozen freeze-dried cells prepared according to this method have been checked for SIMS matrix effects  (7). Ion image quantification has also been achieved in this sample type (8).  
5.3 The procedure described here is amenable to a wide variety of cell cultures and provides a way for studying the response of individual cells for chemical alterations in the state of health and disease and localization of isotopically-labeled molecules and theraputic drugs in cell culture models.
SCOPE
1.1 This guide provides the Secondary Ion Mass Spectrometry (SIMS) analyst with a cryogenic method for analyzing individual tissue culture cells growing in vitro. This guide is suitable for frozen-hydrated and frozen-freeze-dried sample types. Included are procedures for correlating optical, laser scanning confocal and secondary electron microscopies to complement SIMS analysis.  
1.2 This guide is not suitable for cell cultures that do not attach to the substrate.  
1.3 This guide is not suitable for any plastic embedded cell culture specimens.  
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ASTM F813-20(Practice)
Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices

SIGNIFICANCE AND USE
4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical materials and medical devices.  
4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture testing methods have shown good correlation with animal assays when only chemical toxicities are being considered.
Note 1: The results obtained using this method may not predict in vivo behavior which can be influenced by multiple factors such as those arising from site of application or physical properties that may result from design and fabrication.  
4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction of medical devices that are intended to have direct contact with tissue, tissue fluids, or blood. However, care should be taken when testing materials that are absorbable, include an eluting or degradable coating, are liquid or gelatinous in nature, are irregularly shaped solid materials, or have a high density or mass, to make sure that the method is applicable. If leachables from the test sample are capable of diffusing through the agar layer, agarose-based methods such as Test Method F895 may be considered as an alternate method, depending on sample characteristics, or in cases where investigators wish to further evaluate the cytotoxic response of cells underlying the test sample.
SCOPE
1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.  
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods may be compared.  
1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.  
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.  
1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred; only that the L-929 is a well characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement 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.

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ASTM
ASTM E1968-19(Practice)
Standard Practice for Microcrystal Testing in Forensic Analysis for Cocaine

SIGNIFICANCE AND USE
5.1 This technique involves a chemical-precipitation reaction between cocaine and the precipitating reagent. The habit and the aggregation of the crystals formed could be used to distinguish cocaine from other drugs (6).  
5.2 This technique can be utilized on cocaine present in either the salt or free base form.  
5.3 This technique does not distinguish between the salt and free base forms.
SCOPE
1.1 This practice describes procedures applicable to the analysis of cocaine using multiple microcrystal tests (1-6).2  
1.2 These procedures are applicable to cocaine, which is present in solid form or an injectable liquid form. They are not typically applicable to the analysis of cocaine in biological samples.  
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 These procedures could generate observations indicating a positive test for cocaine or its enantiomers which could be incorporated into the analytical scheme as defined by the laboratory.  
1.5 This standard cannot replace knowledge, skills, or abilities acquired through appropriate education, training, and experience (see Practice E2326) and is to be used in conjunction with professional judgment by individuals with such discipline-specific knowledge, skills, and abilities.  
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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ASTM
ASTM E2125-19(Practice)
Standard Practice for Microcrystal Testing in Forensic Analysis for Phencyclidine and Its Analogues

SIGNIFICANCE AND USE
5.1 This technique involves a chemical-precipitation reaction between the phencyclidine or its analogues and the precipitating reagent. The habit and the aggregation of the crystals formed could be used to distinguish phencyclidine or its analogues from other drugs.  
5.2 This technique can be utilized on phencyclidine or its analogues present in either the salt or free base form.  
5.3 This technique does not distinguish between salt and free base forms.
SCOPE
1.1 This practice describes procedures applicable to the analysis of phencyclidine and its analogues using microcrystal tests (1-8).2  
1.2 These procedures are applicable to phencyclidine and its analogues which are present in solid form or in a liquid form. They are not typically applicable to the analysis of phencyclidine and its analogues in biological samples.  
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 These procedures could generate observations indicating a positive test for phencyclidine and its analogues which could be incorporated into the analytical scheme as defined by the laboratory.  
1.5 This standard cannot replace knowledge, skills, or abilities acquired through appropriate education, training, and experience (see Practice E2326) and is to be used in conjunction with sound professional judgment by individuals with such discipline-specific knowledge, skills, and abilities.  
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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