ASTM F2529-13(2021)

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: F2529 − 13 (Reapproved 2021)
Standard Guide for
in vivo Evaluation of Osteoinductive Potential for Materials
Containing Demineralized Bone (DBM)
This standard is issued under the fixed designation F2529; 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 mendations issued by the World Trade Organization Technical
Barriers to Trade (TBT) Committee.
1.1 This guide covers general guidelines to evaluate the
effectiveness of DBM-containing products intended to cause
2. Referenced Documents
and/or promote bone formation when implanted or injected in
2.1 ASTM Standards:
vivo. This guide is applicable to products that may be com-
D1193Specification for Reagent Water
posed of one or more of the following components: natural
D5056Test Method for Trace Metals in Petroleum Coke by
biomaterials (such as demineralized bone), and synthetic bio-
Atomic Absorption
materials (such as calcium sulfate, glycerol, and reverse phase
E508Test Method for Determination of Calcium and Mag-
polymeric compounds) that act as additives, fillers, and/or
nesium in Iron Ores by Flame Atomic Absorption Spec-
excipients (radioprotective agents, preservatives, and/or han-
trometry
dling agents) to make the demineralized bone easier to ma-
F565PracticeforCareandHandlingofOrthopedicImplants
nipulate. It should not be assumed that products evaluated
and Instruments
favorably using this guidance will form bone when used in a
F895TestMethodforAgarDiffusionCellCultureScreening
clinical setting. The primary purpose of this guide is to
for Cytotoxicity
facilitate the equitable comparison of unique bone-forming
F981Practice for Assessment of Compatibility of Biomate-
products in in vivo heterotopic models of osteoinductivity.The
rials for Surgical Implants with Respect to Effect of
purpose of this guide is not to exclude other established
Materials on Muscle and Insertion into Bone
methods.
F1854Test Method for Stereological Evaluation of Porous
1.2 The values stated in SI units are to be regarded as
Coatings on Medical Implants
standard. No other units of measurement are included in this
F2131Test Method forIn Vitro Biological Activity of Re-
standard.
combinant Human Bone Morphogenetic Protein-2
(rhBMP-2) Using the W-20 Mouse Stromal Cell Line
1.3 This standard does not purport to address all of the
F2721Guide for Pre-clinical in vivo Evaluation in Critical
safety concerns, if any, associated with the use of DBM-
Size Segmental Bone Defects
containing bone-forming/promoting products. It is the respon-
sibility of the user of this standard to establish appropriate 2.2 Federal Documents:
safety, health, and environmental practices involved in the 21 CFR 58Good Laboratory Practice for Nonclinical Labo-
development of said products in accordance with applicable ratory Studies
regulatory guidance documents and in implementing this guide 21 CFR 820Quality System Regulation
to evaluate the bone-forming/promoting capabilities of the 21 CFR 1270Human Tissue Intended for Transplantation
product. 21 CFR 1271Human Cells, Tissues, and Cellular and
Tissue-Based Products
1.4 This international standard was developed in accor-
21 CFR 610.12General Biological Products Standards—
dance with internationally recognized principles on standard-
General Provisions—Sterility
ization established in the Decision on Principles for the
Q1E Evaluation of Stability DataFDAGuidance for Indus-
Development of International Standards, Guides and Recom-
try
1 2
This guide is under the jurisdiction ofASTM Committee F04 on Medical and For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Surgical Materials and Devices and is the direct responsibility of Subcommittee contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
F04.44 on Assessment for TEMPs. Standardsvolumeinformation,refertothestandard’sDocumentSummarypageon
Current edition approved Aug. 1, 2021. Published August 2021. Originally the ASTM website.
approved in 2013. Last previous edition approved in 2013 as F2529–13. DOI: Available from Food and DrugAdministration (FDA), 10903 New Hampshire
10.1520/F2529-13R21. Ave., Silver Spring, MD 20993-0002, http://www.fda.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F2529 − 13 (2021)
Container and Closure Integrity Testing in Lieu of Sterility the physical or chemical properties/characteristics of that
Testing as a Component of the Stability Protocol for bone-forming product.
Sterile ProductsFDA Guidance Document
3.1.3 bone—hardconnectivetissueoftheskeletalsystemin
Eligibility Determination for Donors of Human Cells,
vertebrates comprised of collagen, growth factors, and an
Tissues, and Cellular and Tissue-Based ProductsGuid-
inorganicrigidmatrixcontainingcalcium,phosphateandother
ance for Industry
minerals, and various cellular elements, including osteoblasts,
Guidance for the Preparation of a Premarket Notification
osteocytes, osteoclasts, and hematopoietic cells.
Application for a Surgical MeshGuidance for Industry
3.1.3.1 Discussion—Bone is made up of cortical and can-
and/or for FDA Reviewers/Staff and/or Compliance
cellous bone tissue. It serves as the point of attachment for
2.3 AAMI/ISO Documents:
muscles and tendons and is load-bearing.
AAMI TIR 17Compatibility of Materials Subject to Steril-
3.1.4 bone-forming product (containing DBM)—as used in
ization
this guide, a DBM-containing bone-forming product may be
AAMI/ISO 22442-01Medical Devices Utilizing Animal
comprisedofmultiplecomponentsincluding,butnotrestricted
Tissues and Their Derivatives—Part 1: Application of
to, demineralized bone, growth factors, differentiation factors,
Risk Management
osteoprogenitor cells, mesenchymal stem cells, biologically
AAMI/ISO 22442-03Medical Devices Utilizing Animal
active carrier(s), and/or non-biologically active carrier(s).
Tissues and Their Derivatives—Part 3: Validation of the
3.1.5 bone marrow—tissuelocatedinthecancellousportion
Elimination and/or Inactivation of Viruses and Transmis-
and cavities (medullary canal) of most bones.
sible Spongiform Encephalopathy (TSE) Agents
3.1.5.1 Discussion—Bone marrow is highly vascular and
2.4 ANSI/AAMI/ISO Documents:
occurs in two forms: white/yellow marrow, comprised mostly
ANSI/AAMI/ISO 11137Parts 1–3 Sterilization of Health
of adipose cells located primarily in the long bones, and red
Care Products—Radiation
marrow, which primarily produces and contains pluripotent
ANSI/AAMI/ISO 10993Biological Evaluation of Medical
stemscellsandredbloodcells,platelets,andwhitebloodcells
Products
derived from them. In adults, red marrow is located primarily
2.5 ICH Documents:
in the flat bones.
ICH Harmonised Tripartite Guideline Q5CQuality of Bio-
3.1.6 bone tissue—the tissue component of a bone com-
technological Products—Stability Testing of
prised of a mineralized collagenous matrix formed and main-
Biotechnological/Biological Products
tained through the action of osteoblastic cells and osteocytes
ICHHarmonisedTripartiteGuidelineQ5A(R1)ViralSafety
remodeled through the action of osteoclasts.
Evaluation of Biotechnology Products Derived from Cell
Lines of Human or Animal Origin
3.1.7 cartilage—connective tissue that is a major constitu-
2.6 USP Document: ent of the embryonic and young vertebrate skeleton, largely
United States Pharmacopeia Chapter <71>
replaced with bone and bone marrow bone with maturation.
3.1.7.1 Discussion—It is comprised mostly of Type II col-
3. Terminology
lagen and proteoglycans and found in joints, the outer ear,
bronchi, and larynx. There are three major types of cartilage:
3.1 Definitions:
hyaline cartilage, which is adapted for joint surfaces by virtue
3.1.1 additive—ingredientsthatmaybeusedtopreservethe
of its smoothness and ability to withstand compression;
product, provide radioprotection, and/or act as bulk filler
fibrocartilage, found in the outer ear, nose, and meniscus; and
and/or binding agent.
elasticcartilage,foundintheouterearandepiglottis.Cartilage
3.1.1.1 Discussion—Ithasnointendedmodeofaction,such
is also formed by the action of bone morphogenetic protein(s)
as causing cells to transform lineage, once implanted.
(BMPs) in concert with other peptide factors on mesenchymal
3.1.2 biologically active carrier—a component added to a
stem cells.
DBM-containing bone-forming product that results in a physi-
3.1.8 cortical bone—thin superficial layer of compact bone
ological and/or biochemical transformation in the implant site
independentofotherconstituentsinthebone-formingproduct. tissuethatconstitutestheprimaryload-bearingcomponentofa
bone.
3.1.2.1 Discussion—Thetransformationmaybedesirableor
untoward. A biologically active carrier may also contribute to
3.1.9 demineralized bone—bone tissue wherein the average
mineral content, typically measured as calcium, is less than or
equal to 8% by dry weight.
Available from Association for the Advancement of Medical Instrumentation
3.1.9.1 Discussion—As used in this guide, dry weight
(AAMI), 4301 N. Fairfax Dr., Suite 301, Arlington, VA 22203-1633, http://
means lacking significant or measurable residual moisture
www.aami.org.
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
(<5%).
4th Floor, New York, NY 10036, http://www.ansi.org.
3.1.10 differentiation factors—proteins with the ability to
Available from International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use (ICH), ICH
induce or alter cell differentiation and/or proliferation with
Secretariat, c/o IFPMA, 15 ch. Louis-Dunant, P.O. Box 195, 1211 Geneva 20,
subsequent tissue morphogenesis.
Switzerland, http://www.ich.org.
3.1.10.1 Discussion—For example, BMP-2, BMP-4, and
AvailablefromU.S.Pharmacopeia(USP),12601TwinbrookPkwy.,Rockville,
MD 20852-1790, http://www.usp.org. BMP-7(OP-1) are differentiation factors with the ability to
F2529 − 13 (2021)
induce bone and cartilage formation in tissues or implantation 3.1.23.1 Discussion—A carrier may also contribute to the
sites that would otherwise not form bone or cartilage by physical or chemical properties/characteristics of that bone-
causing mesenchymal progenitor cells to differentiate into forming product.
chondrocytes and/or osteoblasts.
3.1.24 nude mouse—a mouse with a genetically deficient
3.1.11 endochondral ossification—process by which bones immune system as the result of a dysfunctional rudimentary
in embryonic and juvenile animals grow while maintaining thymus and thus is deficient in T-cells and incapable of
functional proportions of the skeleton and joints.
producing cytotoxic effector cells and a graft-versus-host
3.1.11.1 Discussion—Bone-forming products considered in response.
this guide mimic this process: post-implantation inflammation
3.1.24.1 Discussion—ThereforetheT-cellresponsetoxeno-
leads to recruitment of mesenchymal stem cells, and the
genic material is abrogated. However, the animals are able to
BMPS/growth factors cause the mesenchymal stems cells to
mount an innate immune response (macrophages, giant cells,
differentiate into chondroblasts, which synthesize cartilage
and granulocytes) and thus are capable of mounting an
matrix. The chondrocytes undergo hypertrophy and calcify
immunological response to non-biocompatible products.
their extracellular matrix. This matrix is then resorbed by
3.1.25 nude rat—a rat with a genetically deficient immune
osteoclasts, angiogenesis occurs, and osteoprogenitor cells
systemastheresultofadysfunctionalrudimentarythymusand
migrate to the calcified cartilage by means of the neovascula-
thus deficient in T-cells and incapable of producing cytotoxic
ture. Osteoblasts synthesize bone on the cartilage scaffold and
effector cells and a graft-versus-host response.
new bone marrow forms, and is incorporated into the new
3.1.25.1 Discussion—ThereforetheT-cellresponsetoxeno-
tissue and ultimately is remodeled.
genic material is abrogated. However, the animals are able to
3.1.12 evidence of endochondral bone formation—
mount an innate immune response (macrophages, giant cells,
chondroblasts,chondrocytes,cartilage,osteoblasts,osteocytes,
and granulocytes) and thus are capable of mounting an
new bone, and bone marrow.
immunological response to non-biocompatible products.
3.1.13 excipient—constituents of a DBM-containing bone-
3.1.26 orthotopic—in the context of this guide, an
forming product that are not the active substance.
implantation/transplantation site within or in direct contact
3.1.14 fascia—fibrous connective tissue between two layers with bone tissue.
of tissues such as muscle, or an internal organ and the
3.1.27 ossicle—a spherical piece of bone consisting of an
abdominal wall.
outer core of cortical bone and an inner region of trabecular
3.1.15 fibrous connective tissue—connective tissue pro- bone and bone marrow.
duced by fibroblasts.
3.1.28 osteoblasts—cells exhibiting the appropriate protein
3.1.16 fillers—an ingredient that may used to expand the
andgeneexpressioncharacteristicsnecessaryfortheformation
volume of the implant material and may possess osteoconduc- and maintenance.
tive properties but does not cause cells to transform lineage
3.1.29 osteoconduction—the ability of a material/bony im-
once implanted.
plant site to support new bone formation by providing an
3.1.17 gelatin capsule—capsule made of gelatin used to environment in which pre-existing osteoid cells capable of
contain materials for implantation.
synthesizing and secreting components essential to the forma-
tion lead to growth of new host bone into/onto the material.
3.1.18 growth factors—proteins produced by cells that
stimulate or inhibit cell proliferation and may affect differen-
3.1.30 osteogenic—indicating the presence of cells capable
tiation.
of synthesizing and secreting components essential to the
formation of bone at some implant site as well as the
3.1.19 heterotopic—in the context of this guide, an
osteoconductive capacity necessary for causing/allowing bone
implantation/transplantation site that is not in direct contact
formation in a tissue or implantation site.
with bone, such as skeletal muscle.
3.1.30.1 Discussion—Additionally, BMPs would also be
3.1.20 intermuscular—within that space occurring between
considered osteogenic as these are some of the components
anatomically identifiable muscles, frequently associated with
secreted by cells that cause the cells to be osteogenic.
little to no bleeding at the time of surgical dissection.
3.1.31 osteoinduction—the ability of a substance to stimu-
3.1.21 intramuscular—within an anatomically identifiable
late cells to differentiate along some osteoprogenitor pathway
bundle of muscle tissue, frequently associated with occurrence
resulting in cells capable of synthesizing and secreting com-
of bleeding at the time of surgical dissection.
ponents essential to the formation of bone at an implant site.
3.1.22 mesenchymal stem cells—cells derived from mesen-
3.1.32 osteoprogenitor cells—cells with the capacity to
chyme that are capable of proliferating and differentiating into
migrate, proliferate, and/or differentiate into osteoblasts under
connectivetissuesuchascartilageandbone,blood,lymphatic,
osteoinductive, osteoconductive, and/or osteogenic conditions.
and blood vessels.
3.1.33 subcutaneous—below the skin and above the fascia
3.1.23 non-biologically active carrier—a component added
covering the underlying tissue.
to a DBM-containing bone-forming product that by itself does
not result in the differentiation or proliferation in the cells 3.1.34 trabecular bone—bone tissue comprised of spicules
infiltrating the implant site. that form a lattice filled with bone marrow.
F2529 − 13 (2021)
3.1.34.1 Discussion—It is also commonly referred to as ride inactivated DBM (1), mineralized cancellous bone and
cancellous bone. Trabecular bone constitutes the secondary heat inactivated DBM (2) and positive controls could be
previously in vivo qualified DBM.
load-bearing component of a bone.
5.2 Athymic (nude) Rodents—Mice (mu/mu) or rats (nu/nu)
4. Significance and Use
with genetically deficient immune systems are the preferred
animal models for assessing bone formation for products
4.1 This guide covers animal implantation methods and
comprised of human demineralized bone in heterotopic sites.
analysis of the explanted DBM-containing material to deter-
Athymicanimalshaveadysfunctionalrudimentarythymusand
mine whether a material or substance possesses osteoinductive
thus are T-cell deficient and incapable of producing cytotoxic
potential,asdefinedbyitsabilitytocausebonetoform in vivo
effector cells and a graft-versus-host response. Therefore, the
at a site that would otherwise not support bone formation, that
T-cell response to xenogenic material is absent. However, the
is,heterotopicallyinaskeletalmuscleimplantsite.For in vitro
animals are able to mount an innate immune response
evaluation see Test Method F2131 for in vitro assessment of
(macrophages, giant cells, and granulocytes) and thus are
rhBMP 2.
capable of having an immunological response to non-
biocompatibleormicrobiologically(culture)positiveproducts.
4.2 The test methods described here may be suitable for
Gender is generally not a factor; however, male athymic
defining product specifications, cGMP lot release testing,
mice/rats should be considered for implantation unless estrous
research evaluation, regulatory submission, and so forth, but a
cycles in female animals have been demonstrated not to
positive outcome should not be presumed to indicate that the
negatively impact the desired studies. In addition, adolescent
product will be osteoinductive in a human clinical application.
mice/rats (six to nine weeks old at the time of implant) are
Atpresent,theonlydirectassaystoassessnewboneformation
recommended as studies have demonstrated that older animals
are in vivo, since the property of bone conduction or induction
may not respond as effectively to osteoinductive materials (3,
can only be assessed in a heterotopic or orthotopic site in a
4). The choice of animal, mouse versus rat, is determined to a
living animal. When these products are implanted in an
large extent by the size (volume) of the material being tested.
orthotopic site, osteogenic factors already present at the im-
5.3 Implant Mass/Volume—In general, implant mass/
plantation site may contribute to and enhance bone formation
volume (DBM+Excipient/additive/filler) of 15 to 120 mg can
in conjunction with the osteoconductive nature of the product.
beconvenientlyassessedusingthenudemouse,whereaslarger
Thus, orthotopic implantation of products may result in bone
samples of 100 to 250 mg are able to be tested in the nude rat
formation by acting on existing bone-forming cells and not by
model. The amount of DBM implanted into a nude mouse
causing mesenchymal stem cells to become osteochondropro-
should ideally be 20 65mg (3, 4) and for implantation into
genitorcells.Incontrast,whentheseproductsareimplantedin
nude rats the amount of DBM should be 40 65mg (1). For
a heterotopic site, no native osteogenic factors are present to
example, a recommended athymic mouse implant may be
contribute to or enhance bone formation. Thus, heterotopic
comprisedof20mgofDBMand60mgofexcipientforatotal
implantationofproductswillonlyresultinnewboneformation
sample size of 80 mg. It is recommended that the
by causing mesenchymal stem cells to become osteochondro-
DBM+excipient groups contain the same amount of bone
progenitor cells. In vitro assays have been described and some
implanted as negative and positive controls so the results are
believe they may correlate to the results obtained from in vivo
comparable and the potential effects of the excipient on
assays.Howeversuch in vitroassaysmeasureonlysomeofthe
osteoinductive potential can be determined.
biochemical marker(s) associated with in vivo bone formation
5.4 Implant Numbers/Sample Size:
and are therefore only indirect assays for osteoinductive
5.4.1 In order to achieve a statistically valid outcome for
activity or the capacity to promote new bone formation. Many
histomorphometry, a minimum of two animals (nude mouse/
factors or combinations of factors contribute to osteoblast
rat)eachwithtwoimplantsperlotarenecessary.Thisachieves
progenitor cells differentiating and/or proliferating into bone-
83%poweratthe95%confidenceinterval.Thisallowsforthe
forming cells in vitro that are both osteoinductive and osteo-
detection of a 12% difference in new bone formation. The
conductive when they are implanted in vivo. Thus, only an in
following is assumed: The percent new bone is 40%; the
vivo assay method currently directly considers the many
standard deviation is 5%; and the alpha is 0.05.
potential factors involved in new bone formation induced by
5.4.2 A minimum of four implants in four different nude
DBM-containing biomaterials. The qualification of a DBM or
rats, for a total of 16 implants per test group, or a minimum of
DBM-containingmaterialshouldalsoencompassproductchar- two implants in six different nude mice, for a total of twelve
acterization such as that described in Appendix X1. implants per test group, may be required to achieve statistical
significance where product variability and performance are
uncertain (1, 3, 4). However, each investigator will need to
5. Animal Models
perform and report his/her own statistical assessment to ac-
5.1 General Note—Appropriate positive and negative con-
count for the variability or product/process change under
trols shall be used for research purposes and during qualifica-
investigation.
tion of new test articles into a validated system, but are not
required for routine lot release testing once validated. For 8
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
example, negative controls could be 4M guanidine hydrochlo- this standard.
F2529 − 13 (2021)
5.5 Heterotopic Implant Locations: strated to possess osteoinductive potential in vivo), to demon-
5.5.1 The preferred heterotopic test site is in the animal’s strate that the test system is unaffected by the implant
methodologies, anesthetizing agents and analgesics.
skeletalmuscle.Intermuscularpouchesintheglutealmuscleor
biceps femoris are some appropriate implant sites for the
6.3 It is recommended that non-steroidal anti-inflammatory
athymic mouse model (3, 4). Intermuscular pouches in the
drugs be avoided because of potential confounding effects.
gluteal, biceps femoris, pectoralis, abdominal, or dorsal
6.4 A minimum of two animals, each receiving two im-
muscles are some appropriate implant sites for the athymic rat
plants per lot of test article are recommended.
model (1). Other sites can be qualified using this guide. Care
should be taken that the implant does not touch or rest against
6.5 The in-life portion of the validation shall be 28 to 36
aboneasthiswillprovideosteogenicfactorstotheimplantand days. Going longer than this time frame makes it difficult to
will bias the data towards a false positive. As a general rule,
compare results between laboratories, even with appropriate
intermuscular implantation is one in which minimal bleeding controls. A longer in-life duration should be well justified.
occurs at the site of implantation because the implant goes
6.6 Recommended explant preparation is described in 7.7.
between the fascia. An intramuscular implant is one in which
6.7 DBM to be used a negative control may be inactivated
bleeding occurs because the implant goes between the muscle
using 4M guanidine hydrochloride or heat (2).
fiberswithinamusclebundlesuchthatthebloodoftheanimal
mixes directly with the implanted material at the time of
6.8 Assessments of osteoinductive potential for the valida-
implantation if care is not taken to allow the bleeding to stop tion may be carried out using quantitative histological analysis
and the blood reasonably cleared from the implant site.
such as described in 8.3.
Implantation of DBM intra- versus intermuscularly signifi-
6.9 Acceptance Criteria:
cantly affects the amounts of new bone formed from the same
6.9.1 Elements of new bone formation include:
implant DBM (3). Therefore, to obtain the most objective
chrondroblasts/cytes, osteoblasts/cytes, cartilage, osteoid, new
result from this intentionally ectopic model, the intermuscular
bone, and bone marrow. Percent new bone: 75% of the
implantation is the recommended implant methodology de-
explantsperlotofDBMmustdemonstrate≥10%averagenew
scribed in 7.8.4, thus the amount of blood in the implant site
boneformation.Byrequiring75%oftheexplantstoformnew
should be minimized so as not to significantly bias the results.
bone it demonstrates the intra-lot consistency of each batch/lot
Minimizing blood in the implant site allows for a more
tested.
accurate assessment of the osteoinductive potential of the
6.9.2 The percent is arrived at by the calculation in 8.3.16.
product;howeverduringclinicaluse,itisrecognizedthatthere
6.9.3 All negative controls should demonstrate <10% new
willbebloodintheimplantsiteaswellasboneandosteogenic
bone formation.
components.Implantsmayalsobeplacedsubcutaneouslyover
6.10 Recommended Minimum Protocol/Report Contents:
thepectoralismusclesofthechestintheathymicrat;however,
6.10.1 Title, date, purpose, cGLP statement, sponsor name
subcutaneous sites have proven to be less favorable in their
and address, test facility name and address, test article charac-
ability to support osteoinduction in the nude mouse (3).
terization statement/disclaimer.
Furthermore, a false negative result is more probable with a
6.10.2 Justification for the test system, description of test
subcutaneousimplantmodelthanwiththemuscleimplantation
system (species, gender, age, source, weight range, number of
model due to the different embryonic origin of these two
animals involved in the protocol, animal identification, Insti-
tissues.
tutional Animal Care and use Committee (IACUC) protocol
5.5.2 For cGMP lot release testing and testing to support
number, and approval date), husbandry (receipt, acclimation,
regulatory submissions, validated implantation methodologies,
housing, environment, diet, water, and USDA animal welfare
testandassessmentsystemsshallbeemployed.Inaddition,the
act statement).
test article shall be qualified into the validated testing system.
6.10.3 Study design, study test method, test article
Thequalificationshouldencompassproductcharacterizationas
preparation, test article administration, health observations,
described in Appendix X1 and the successful implantation of
termination, histopathology analysis, interpretation of results,
three separate lots of product into a minimum of two athymic
control of bias, data analysis, statistical methods, assay
mice/rats, each receiving a minimum of two implants (as
validity,acceptancecriteria,protocolchanges,recordretention,
detailed above).
references, compliance statements (GLP and AAALAC), test
article identification, and test articles disposition.
6. Method Validation and New Product Qualification into
6.10.4 Report should contain any protocol deviations.
a Validated Model System
6.10.5 Signature and date of responsible parties performing
6.1 The validation shall be performed in accordance with
and requesting the validation.
cGLPrequirements 21 CFR Part 58. The test system (athymic
mouse/rat) and procedures shall be validated prior to specific
7. Recommended Implant Methodologies
bone-forming product testing.
7.1 Sample Preparation—All implant volumes should be
6.2 Validation should involve the implantation of a mini- normalized to contain equal amounts of DBM as measured by
mum of three different lots of DBM, a negative control (for freeze-dried weight. Implants for athymic mice should contain
example, inactivated DBM or mineralized cancellous bone), 20 6 5 mg of DBM, and 40 6 5 mg for athymic rats (3, 4).
and a positive control (for example, DBM previously demon- Other implant volumes can be utilized but positive and
F2529 − 13 (2021)
negative controls should be of equivalent weight and th
...