ASTM E2525-22

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.
Designation: E2525 − 22
Standard Test Method for
Evaluation of the Effect of Nanoparticulate Materials on the
Formation of Mouse Granulocyte-Macrophage Colonies
This standard is issued under the fixed designation E2525; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method provides a protocol for quantitative
F1903Practice for Testing for Cellular Responses to Par-
analysis of the effect of nanoparticulate materials in physi-
ticles in vitro
ologic solution (isotonic, pH 7.2 6 0.2) on granulocyte-
2.2 ANSI Standard:
macrophage colony-forming units (CFU-GM).
ANSI/ AAMI ST72 Bacterial Endotoxins—Test
1.2 CFU-GM reflects the number of viable bone marrow
Methodologies, Routine Monitoring, and Alternatives to
cellswhichdifferentiateintogranulocytesandmacrophages.A
Batch Testing
decrease in CFU-GM count is indicative of a test substance’s
3. Terminology
toxicity to bone marrow and is commonly used for the
identification of drug products with myelosuppressive
3.1 Abbreviations:
properties, a form of immunosuppression.
3.1.1 CFU-GM—colony forming unit of granulocyte and
macrophage
1.3 This test method employs murine bone marrow he-
3.1.2 DMSO—dimethyl sulfoxide
matopoietic stem cells which proliferate and differentiate to
3.1.3 DPBS—Dulbecco’s phosphate buffered saline
form discrete cell clusters or colonies which are counted.
3.1.4 FBS—fetal bovine serum
1.4 This test method is part of the in vitro preclinical
3.1.5 IMDM—Iscove’s media
characterization cascade for nanoparticulate materials for sys-
temic administration in medical applications. 3.1.6 LPS—ipopolysaccharide
1.5 The values stated in SI units are to be regarded as
4. Summary of Test Method
standard. No other units of measurement are included in this
4.1 The effect of nanoparticulate materials on the formation
standard.
of granulocyte and macrophage colonies is assessed. Bone
1.6 This standard does not purport to address all of the
marrow cells are obtained from mice and cultured in stimula-
safety concerns, if any, associated with its use. It is the tory media. The number of colony forming units following
responsibility of the user of this standard to establish appro- contactwithnanoparticlesiscountedvisuallyandcomparedto
baseline and positive control. This determines if the nanopar-
priate safety, health, and environmental practices and deter-
ticulate material in physiologic solution is stimulatory or
mine the applicability of regulatory limitations prior to use.
inhibitory to bone marrow stem cells. Aseptic procedures are
1.7 This international standard was developed in accor-
necessary.
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
5. Significance and Use
Development of International Standards, Guides and Recom-
5.1 Stemcellsofhematopoieticoriginarepluripotentialand
mendations issued by the World Trade Organization Technical
may be particularly sensitive to the effects of stimulation by
Barriers to Trade (TBT) Committee.
nanoparticulate materials.
1 2
This test method is under the jurisdiction of ASTM Committee E56 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Nanotechnology and is the direct responsibility of Subcommittee E56.03 on contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Environment, Health, and Safety. Standards volume information, refer to the standard’s Document Summary page on
CurrenteditionapprovedJuly1,2022.PublishedJuly2022.Originallyapproved the ASTM website.
in 2008. Last previous edition approved in 2013 as E2525 – 08(2013), which was Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
withdrawn in April 2022 and reinstated July 2022. DOI: 10.1520/E2525-22. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2525 − 22
5.2 The effect of particles on macrophage responses has an 6.3.14 Biohazard safety cabinet approved for level II han-
extensive history and can be assessed by Practice F1903. The dling of biological material.
test method described here will assess the effect on stem cells
6.3.15 Inverted microscope.
which can be progenitor cells to the macrophage line.
6.3.16 Vortex.
6.3.17 Hemocytometer.
6. Reagents and Materials
6.4 Animals—MiceofthestrainC56BL/6,malesorfemales
6.1 Purity of Reagents—Reagent grade chemicals shall be
8weeks to 12 weeks old, are used. Use of the pooled cells
used in all tests. Unless otherwise indicated, it is intended that
derived from at least two (2) animals is highly recommended.
all reagents conform to the specifications of the Committee on
NOTE 2—The source of the equipment, where given, is shown for
Analytical Reagents of theAmerican Chemical Society where
information purposes only to aid laboratories initiating this procedure.
such specifications are available. Other grades may be used,
Equivalent equipment from other suppliers may be used.
provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of
7. Procedure
the determination.
7.1 Aseptic Precautions are required.
6.2 Reagents and Supplies (see Note 1):
7.2 Reagent and Control Preparation:
6.2.1 MethoCult medium, (optimized for growth and enu-
7.2.1 MethoCult Medium—The MethoCult medium is sup-
meration of granulocyte-macrophage progenitor cells) Stem-
plied in 100mL size batches. It is recommended by the
Cell Technologies Inc cat.# 03534.
manufacturer that the medium be thawed at room temperature
6.2.2 FBS prescreened for hematopoietic stem cells, Stem-
or in a refrigerator overnight, vortexed to mix the ingredients,
Cell Technologies Inc cat.# 06200.
then left at a room temperature for approximately 5 min to
6.2.3 IMDM with 2% FBS, StemCell Technologies Inc
allowairbubblestodissipate.Ifusinganothersourceofmedia,
cat.# 07700.
follow the instructions indicated by the supplier. Use a 16-
6.2.4 Sterile distilled water.
gauge blunt-end needle to dispense 3mL aliquots of the
6.2.5 Cisplatin, [cis-diammineplatinum(II) dichloride],
MethoCult medium into sterile 15mL tubes. Store the ali-
(positive control) Sigma-Aldrich cat# P4394.
2+ 2+
quoted medium at a nominal temperature of –20°C. Before the
6.2.6 Sterile Ca /MG -free DPBS, (negative control)
test,thawtherequirednumberoftubesatroomtemperaturefor
Sigma-Aldrich cat.# D8537.
approximately 20 min and then keep on ice prior to use.
NOTE 1—The source of the reagents is shown for information purposes
Repeated freezing and thawing should be avoided.
onlytoaidlaboratoriesinitiatingthisprocedure.Equivalentreagentsfrom
7.2.2 50 mmol/L Cisplatin (Positive Control)—Cisplatin is
other suppliers may be used.
supplied in a lyophilized form. Reconstitute the lyophilized
6.3 Equipment (see Note 2)—Aseptic procedures are neces-
powder by adding the appropriate amount of DMSO to make a
sary and care should be used in acquiring sterile equipment as
stock solution with nominal concentration of 50 mmol/L.
needed.
Prepare small aliquots and store at a nominal temperature of
6.3.1 Pipettes covering the range of 0.05mL to 10 mL.
–80 °C. Prior to use in the assay, thaw an aliquot of the stock
6.3.2 35-mmculturedishesprescreenedtosupportstemcell
solution at room temperature and dilute in IMDM supple-
growth and differentiation, StemCell Technologies Inc cat.#
mented with 2% FBS to bring the Cisplatin concentration to 2
27100.
mmol/L. One hundred fifty (150) µL of this intermediate
6.3.3 Blunt-end 16-gauge needles, StemCell Technologies
solution is then added to 3 mL of culture medium. Final
Inc cat.# 03534.
concentrationofCisplatininthepositivecontrolsampleis100
6.3.4 100-mm Petri dishes.
µmol/L.
6.3.5 Plastic beakers.
7.3 Preparation of Study Samples:
6.3.6 Polypropylene tubes 50mL and 15mL.
7.3.1 This assay requires 1200 µL of nanoparticles, 150 µL
6.3.7 Centrifuge.
samples in duplicate for each of four (4) concentrations. The
6.3.8 Refrigerator, 2°C to 8°C.
nanoparticles subjected to the biological test environment
6.3.9 Freezer, –20°C.
should have been characterized as appropriate to allow ad-
6.3.10 Cell culture incubator with 5% CO and 95%
equate data interpretation and to help provide information to
humidity.
predict biological responses. For example, lot-to-lot variations
6.3.11 CO euthanasia box, or appropriate equipment ap-
inparticlesizeandsurfacecharacteristicsoftheparticlescould
proved by institution.
result in different assay results. For this assay, the particles
6.3.12 Scissors for tissue dissection.
shall be provided in physiologic solution (isotonic with pH
6.3.13 Forceps.
7.2 60.2) and this solution shall be defined. The preparation
shall be sterile and the level of LPS provided or determined by
the testing laboratory.ANSI/AAMI ST72 may be helpful. The
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
number of particles/mL and mg/mL shall be indicated.
listed by the American Chemical Society, see Analar Standards for Laboratory
7.3.2 The test sample shall be used at the highest concen-
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
tration possible
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