ASTM F1439-03(2013)
(Guide)Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
Standard Guide for Performance of Lifetime Bioassay for the Tumorigenic Potential of Implant Materials
SIGNIFICANCE AND USE
4.1 This guide is not intended to specify the exact method of conducting a test for any particular material but only to present some of the criteria that should be considered in method design and possible problems that could lead to misleading results. In the development of the actual test protocol, it is recommended that recognized tumorigenesis bioassay procedures be consulted.
4.2 The recommendations given in this guide may not be appropriate for all applications or types of implant materials. These recommendations should be utilized by experienced testing personnel in conjunction with other pertinent information and the requirements of the specific material application.
SCOPE
1.1 This guide is intended to assist the biomaterials testing laboratory in the conduct and evaluation of tumorigenicity tests to evaluate the potential for new materials to evoke a neoplastic response. The procedure is generally reserved only for those materials which have not previously been used for human implantation for a significant period of time.
1.2 Assessment of tumorigenicity is one of several procedures employed in determining the biological response to a material as recommended in Practice F748. It is assumed that the investigator has already determined that this type of testing is necessary for a particular material before consulting this guide. The recommendations of Practice F748 should be considered before a study is commenced.
1.3 Whenever possible, it is recommended that a battery of genotoxicity procedures be initiated and proposed as an alternative to an in-vivo tumorigenicity bioassay. Genotoxicity assays may also be considered as initial screening procedures due to the sensitivity of the assays, the significant reduction in time to gain valuable data, and the desire to reduce the use of animals for testing. Genotoxicity assays that may be considered are outlined in Guides E1262, E1263, E1280, and E2186, and Practices E1397 and E1398. Additionally, other genotoxicity testing which might be considered (but which do not yet have ASTM test methods) include Salmonella/Mammalian-Microsomal Plate Incorporation Mutagenicity Assay, In Vivo Cytogenetics Bone Marrow Chromosomal Damage Assay, BALB/3T3 Morphological Transformation of Mouse Embryo Cells, and the Mouse Micronucleus Assay. The investigator is advised to consider carefully the appropriateness of a particular method for his application after a review of the published literature.
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.
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Designation: F1439 − 03 (Reapproved 2013)
Standard Guide for
Performance of Lifetime Bioassay for the Tumorigenic
Potential of Implant Materials
This standard is issued under the fixed designation F1439; 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 priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
1.1 This guide is intended to assist the biomaterials testing
laboratoryintheconductandevaluationoftumorigenicitytests
2. Referenced Documents
toevaluatethepotentialfornewmaterialstoevokeaneoplastic
2.1 ASTM Standards:
response. The procedure is generally reserved only for those
E1262 Guide for Performance of Chinese Hamster Ovary
materials which have not previously been used for human
Cell/Hypoxanthine Guanine Phosphoribosyl Transferase
implantation for a significant period of time.
Gene Mutation Assay
1.2 Assessment of tumorigenicity is one of several proce-
E1263 Guide for Conduct of Micronucleus Assays in Mam-
dures employed in determining the biological response to a
malian Bone Marrow Erythrocytes
material as recommended in Practice F748. It is assumed that
E1280 Guide for Performing the Mouse Lymphoma Assay
the investigator has already determined that this type of testing
for Mammalian Cell Mutagenicity
is necessary for a particular material before consulting this
E1397 Practice forIn Vitro Rat Hepatocyte DNA Repair
guide. The recommendations of Practice F748 should be
Assay
considered before a study is commenced.
E1398 Practice forIn Vivo Rat Hepatocyte DNA Repair
1.3 Whenever possible, it is recommended that a battery of
Assay
genotoxicity procedures be initiated and proposed as an alter-
E2186 Guide for Determining DNA Single-Strand Damage
native to an in-vivo tumorigenicity bioassay. Genotoxicity
in Eukaryotic Cells Using the Comet Assay
assays may also be considered as initial screening procedures
F748 PracticeforSelectingGenericBiologicalTestMethods
due to the sensitivity of the assays, the significant reduction in
for Materials and Devices
time to gain valuable data, and the desire to reduce the use of
2.2 Other Documents:
animals for testing. Genotoxicity assays that may be consid-
National Toxicology Program General Statement of Work
ered are outlined in Guides E1262, E1263, E1280, and E2186,
fortheConductofToxicityandCarcinogenicityStudiesin
and Practices E1397 and E1398. Additionally, other genotox-
Laboratory Animals
icity testing which might be considered (but which do not yet
OECD Guidelines for Testing of Chemicals: Guideline 451,
have ASTM test methods) include Salmonella/Mammalian-
Carcinogenicity Studies
Microsomal Plate Incorporation Mutagenicity Assay, In Vivo
OECD Guidelines for Testing of Chemicals: Guideline 453,
Cytogenetics Bone Marrow Chromosomal Damage Assay,
Combined Chronic Toxicity/Carcinogenicity Studies
BALB/3T3 Morphological Transformation of Mouse Embryo
Good Laboratory Practice for Nonclinical Laboratory Stud-
Cells, and the Mouse Micronucleus Assay. The investigator is
ies
advisedtoconsidercarefullytheappropriatenessofaparticular
method for his application after a review of the published
literature.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
1.4 This standard does not purport to address all of the contact ASTM Customer Service at service@astm.org. For Annual Book ofASTM
Standards volume information, refer to the standard’s Document Summary page on
safety concerns, if any, associated with its use. It is the
the ASTM website.
responsibility of the user of this standard to establish appro-
Available from National Institute of Environmental Health Sciences, 111 T. W.
Alexander Drive, Research Triangle Park, NC, August 1988. http://
www.niehs.nih.gov/.
1 4
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Available from Organization for Economic Cooperation and Development,
Surgical Materials and Devices and is the direct responsibility of Subcommittee 2001 L Street, N.W., Suite 650, Washington, D.C. 20036-4922. http://
F04.16 on Biocompatibility Test Methods. www.oecd.org/washington/contact.htm.
Current edition approved Oct. 1, 2013. Published October 2013. Originally Available from 21 CFR, Part 58, U.S. Government Printing Office Superinten-
approved in 1992. Last previous edition approved in 2008 as F1439 – 03 (2008). dent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC
DOI: 10.1520/F1439-03R13. 20401, http://www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1439 − 03 (2013)
3. Terminology device will ultimately be used only in the male or female, only
one sex may need to be evaluated. Otherwise, both sexes
3.1 Definitions of Terms Specific to This Standard:
should be used.
3.1.1 carcinogenic—a substance is considered to be carci-
5.4 The decision to use other species for study should be
nogenicifitcanbeshowntobecausallyrelatedtoanincreased
carefully documented in terms of a clear need. The use of
incidence of malignant neoplastic formation.
species which have not previously been used may reduce the
3.1.2 maximum implantable dose—the maximum weight or
amount of comparative data available on control animals.
volume of the test article which can be reasonably implanted
Typical tumor rates for hamsters, rats, and mice have been
into the test site taking into account the gross distention of
tabulated and are available in Refs. (1, 2, 3).
tissue which can occur and its possible effects on test results.
6. Selection of Size and Form of Implant
3.1.3 mutagenic—a substance is said to be mutagenic if it
induces alterations in the genetic code of the cell.
6.1 Tumorigenicity bioassays have traditionally been per-
formed using chemical substances as the challenge. The
3.1.4 tumorigenic—a substance is said to be tumorigenic if
evaluation of implant materials requires that solid material be
it can be shown to be causally related to an increased incidence
implanted in some form. It is important to realize that the
of neoplastic formation whether malignant or benign.
down-sized implants necessary for use in animals will have a
greatersurfaceareatovolumeratio,andthisdifferencemustbe
4. Significance and Use
considered in experimental design.
4.1 Thisguideisnotintendedtospecifytheexactmethodof
6.2 It may be important to determine the site of administra-
conducting a test for any particular material but only to present
tion of the test material that is most appropriate to the end use
someofthecriteriathatshouldbeconsideredinmethoddesign
before determining implant size. The site of implantation
and possible problems that could lead to misleading results. In
should be the paravertebral muscle unless the size of the
the development of the actual test protocol, it is recommended
implant causes this site to be unacceptable. Alternatively, the
that recognized tumorigenesis bioassay procedures be con-
site of implantation should mimic the anticipated end use, if
sulted.
possible. Where a specific material may be utilized in more
4.2 The recommendations given in this guide may not be
than one type of device, multiple sites of administration should
appropriate for all applications or types of implant materials.
be considered if different types of tissue will be contacted. (For
These recommendations should be utilized by experienced
instance, materials that may be in contact with bone or
testing personnel in conjunction with other pertinent informa-
implanted into internal organ tissue might be tested in both
tion and the requirements of the specific material application.
tissues.)
6.3 It should be recognized that the response of the test
5. Choice of Animal Model
animal to an extract of a material may not fully represent the
5.1 These types of bioassays for chemical substances have
response that might be seen if the material itself were to be
traditionally been performed in mice or rats, or both, because implanted. In general, an extract should not be used as a
of their small size, relative cost factors, and lifespan. For the substitute for the actual material of interest.
testing of biomaterials, mice are not recommended because the
6.4 The physical form of the test material should be repre-
small animal size is not conducive to the placement of solid
sentative of that intended for use in human patients and should
implants. The investigator should seriously consider the use of
consider potential material debris, if appropriate. The investi-
one of the traditional models in order to draw upon the
gator should be aware that tests have shown (4) that powdered
extensive information available about typical tumor formation
polymeric materials may not elicit a tumorigenic response
rates and sites in control animals. The National Toxicology
subcutaneously even when prepared from polymers that do
Program recommends the use of Fischer 344 (F344/N) rats.
induce tumors when implanted in the form of a film. The
However, other readily available species and strains may also
impact of physical form and surface properties on tumorigen-
be acceptable for the performance of these studies. Other rat
esis must be carefully considered, in making decisions about
species which have been recommended include Sprague-
the physical form of the implants (5, 6, 7, 8, 9, 10).
Dawley, Long-Evans, and Wistar. Some investigators have
6.5 Researchers have found that the aspect ratio (length/
recommendedtheuseofLong-EvansorWistarRatsbecauseof
diameter) of fiber materials may play a role in the tumorigen-
the difficulty of achieving a two-year lifespan for Fischer and
esis of a particular material (11, 12). When new fibrous
Sprague-Dawley rats.
materials are being tested, the actual fiber length to be
5.2 The currently accepted level of testing in a particular
anticipated in practice should be studied. If fragmentation can
site of implantation or medical specialty should be carefully
be anticipated or is a worse case possibility, an attempt should
researched and regulatory requirements determined before a
be made to document a clinically relevant fiber length.
study design is finalized to ensure acceptability of the final
6.6 The material to be tested should originate from
results.
sample(s) representative of all processing including surface
5.3 The appropriate choice of male or female animals or a
combination should be carefully considered in light of the
The boldface numbers in parentheses refer to the list of references at the end of
particular material and application being investigated. If the this guide.
F1439 − 03 (2013)
finishing,passivation,andsterilizationorotherfinalprocessing experimentcanbeperformedwithfewerthantheusualnumber
that will occur to a finished device. of animals per group, that fact should be documented and the
study design should proceed accordingly.
6.7 Dosage:
6.7.1 Inmostmaterials,theratiobetweenthesurfaceareaof
9. Duration of Study
the implant and the body weight of the animal or person will
9.1 Recommended durations for evaluation of tumorigenic-
have an effect on the amount of extractable substances (if any)
ity in rats is two years.
which leach out of the material. The total weight or volume of
material used in each animal should be in excess of the
9.2 Depending upon the material being evaluated, the early
anticipated dosages to be seen in clinical practice when
results may suggest that the study can be terminated earlier
calculated based upon the ratio of surface area of sample to
than two years without compromising the validity of the study.
body weight of the animal. Consideration should be given to
Examples might include studies in which a significantly
using the maximum implantable dose as the dosage or as one
increased rate of tumor formation or toxicity is being seen in
of multiple dosage levels. For the special case of degradable
the test animals or in one or more dosage groups.
materials, the sample size should be calculated based on the
9.3 Attheterminationofthestudy,amajorityoftheanimals
ratio of sample weight to animal body weight.
in each group should have survived for euthanasia or been
6.7.2 Whenever possible, more than one exposure level
terminated early for study-related reasons such as increased
should be considered to evaluate a dose-response effect.
tumor incidence, spontaneous tumors, or toxicity of the test
7. Choice of Control article. It is expected that a minimum of 50 % of the animals
per sex and per group should survive until final study termi-
7.1 Control groups for this type of study will usually consist
nation barring the above reasons. Moreover, the number of
of identical animals that have not received an implant of the
survivors or study-related terminations should be sufficient for
test material but have been subjected to the remainder of the
detection of effects at the p < 0.05 level of significance. If
surgical procedures. Additional groups such as housing (ani-
attrition is occurring due to reasons which cannot be attributed
mals which receive no treatment but are housed with the test
to the test articles or spontaneous tumor formation, other
animals) and reference control groups may be included in the
factors should be considered such as environmental and food
study design.
and water problems. This type of attrition can adversely affect
7.2 The investigator should consider a negative control
the validity of a study and the investigator should be cognizant
group in addition to the sham or untreated controls. These
of the importance of prompt investigation of attrition in animal
animals would receive an implant or treatment identical to the
numbers.
test animals but the implant would be manufactured from a
selected negative reference material. This group would then
10. Housing and Postoperative Care
serve to isolate any results due to the implant trauma or
10.1 The animals shall be housed and care provided in
mechanically induced changes.
accordance with the Guide for the Care and Use of Laboratory
8. Size of Test Groups
Animals(13) or other appropriate guidelines.
8.1 The test group and the control group should each
10.2 In addition to the requirements for humane treatment
contain enough animals which will be scheduled to survive to
of animals in 10.1, the facilities and environment used, as well
the end of the study to allow statistically valid conclusions to as any postoperative drug therapies or other treatment
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