ASTM F1439-92(1996)

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: F 1439 – 92 (Reapproved 1996)
Standard Guide for
Performance of Lifetime Bioassay for the Tumorigenic
Potential of Implant Materials
This standard is issued under the fixed designation F 1439; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope E 1262 Guide for the Performance of the Chinese Hamster
Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Trans-
1.1 This guide is intended to assist the biomaterials testing
ferase Gene Mutation Assay
laboratory in the conduct and evaluation of tumorigenicity tests
E 1280 Guide for Performing the Mouse Lymphoma Assay
to evaluate the potential for new materials to evoke a neoplastic
for Mammalian Cell Mutagenicity
response. The procedure is generally reserved only for those
E 1397 Practices for the In-Vitro Rat Hepatocyte DNA
materials which have not previously been used for human
Repair Assay
implantation for a significant period of time.
E 1398 Practices for the In-Vivo Rat Hepatocyte DNA
1.2 Assessment of tumorigenicity is one of several proce-
Repair Assay
dures employed in determining the biological response to a
F 748 Practice for Selecting Generic Biological Test Meth-
material as recommended in Practice F 748. It is assumed that
ods for Materials and Devices
the investigator has already determined that this type of testing
2.2 Other Documents:
is necessary for a particular material before consulting this
National Toxicology Program General Statement of Work
guide. The recommendations of Practice F 748 should be
for the Conduct of Toxicity and Carcinogenicity Studies in
considered before a study is commenced.
Laboratory Animals
1.3 Whenever possible, it is recommended that a battery of
OECD Guidelines for Testing of Chemicals: Guideline 451,
genotoxicity procedures be initiated and proposed as an alter-
Carcinogenicity Studies
native to an in-vivo tumorigenicity bioassay. Genotoxicity
OECD Guidelines for Testing of Chemicals: Guideline 453,
assays may also be considered as initial screening procedures
Combined Chronic Toxicity/Carcinogenicity Studies
due to the sensitivity of the assays, the significant reduction in
Good Laboratory Practice for Nonclinical Laboratory Stud-
time to gain valuable data, and the desire to reduce the use of
ies
animals for testing. Genotoxicity assays that may be consid-
ered are outlined in Guides E 1262 and E 1280 and Practices
3. Terminology
E 1397 and E 1398. Additionally, other genotoxicity testing
3.1 Definitions of Terms Specific to this Standard:
which might be considered (but which do not yet have ASTM
3.1.1 carcinogenic—a substance is considered to be carci-
test methods) include Salmonella/Mammalian-Microsomal
nogenic if it can be shown to be causally related to an increased
Plate Incorporation Mutagenicity Assay, In Vivo Cytogenetics
incidence of malignant neoplastic formation.
Bone Marrow Chromosomal Damage Assay, BALB/3T3 Mor-
3.1.2 maximum implantable dose—the maximum weight or
phological Transformation of Mouse Embryo Cells, and the
volume of the test article which can be reasonably implanted
Mouse Micronucleus Assay. The investigator is advised to
into the test site taking into account the gross distention of
consider carefully the appropriateness of a particular method
tissue which can occur and its possible effects on test results.
for his application after a review of the published literature.
3.1.3 mutagenic—a substance is said to be mutagenic if it
1.4 This standard does not purport to address all of the
induces alterations in the genetic code of the cell.
safety concerns, if any, associated with its use. It is the
3.1.4 tumorigenic—a substance is said to be tumorigenic if
responsibility of the user of this standard to establish appro-
it can be shown to be causally related to an increased incidence
priate safety and health practices and determine the applica-
of neoplastic formation whether malignant or benign.
bility of regulatory limitations prior to use.
2. Referenced Documents
Annual Book of ASTM Standards, Vol 11.05.
2.1 ASTM Standards:
Annual Book of ASTM Standards, Vol 13.01.
Available from National Institute of Environmental Health Sciences, Research
Triangle Park, NC, August 1988.
1 5
This guide is under the jurisdiction of ASTM Committee F-4 on Medical and Available from Organization for Economic Cooperation and Development,
Surgical Materials and Devices and is the direct responsibility of Subcommittee Washington, DC.
F04.16 on Biocompatibility Test Methods. Available from 21 CFR, Part 58, U.S. Government Printing Office, Washington,
Current edition approved Oct. 15, 1992. Published December 1992. DC.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
F 1439
4. Significance and Use down-sized implants necessary for use in animals will have a
greater surface area to volume ratio, and this difference must be
4.1 This guide is not intended to specify the exact method of
considered in experimental design.
conducting a test for any particular material but only to present
6.2 It may be important to determine the site of administra-
some of the criteria that should be considered in method design
tion of the test material that is most appropriate to the end use
and possible problems that could lead to misleading results. In
before determining implant size. The site of implantation
the development of the actual test protocol, it is recommended
should be the paravertebral muscle unless the size of the
that recognized tumorigenesis bioassay procedures be con-
implant causes this site to be unacceptable. Alternatively, the
sulted.
site of implantation should mimic the anticipated end use, if
4.2 The recommendations given in this guide may not be
possible. Where a specific material may be utilized in more
appropriate for all applications or types of implant materials.
than one type of device, multiple sites of administration should
These recommendations should be utilized by experienced
be considered if different types of tissue will be contacted. (For
testing personnel in conjunction with other pertinent informa-
instance, materials that may be in contact with bone or
tion and the requirements of the specific material application.
implanted into internal organ tissue might be tested in both
5. Choice of Animal Model tissues.)
6.3 It should be recognized that the response of the test
5.1 These types of bioassays for chemical substances have
animal to an extract of a material may not fully represent the
traditionally been performed in mice or rats, or both, because
response that might be seen if the material itself were to be
of their small size, relative cost factors, and lifespan. For the
implanted. In general, an extract should not be used as a
testing of biomaterials, mice are not recommended because the
substitute for the actual material of interest.
small animal size is not conducive to the placement of solid
6.4 The physical form of the test material should be repre-
implants. The investigator should seriously consider the use of
sentative of that intended for use in human patients and should
one of the traditional models in order to draw upon the
consider potential material debris, if appropriate. The investi-
extensive information available about typical tumor formation
gator should be aware that tests have shown (4) that powdered
rates and sites in control animals. The National Toxicology
polymeric materials may not elicit a tumorigenic response
Program recommends the use of Fischer 344 (F344/N) rats.
subcutaneously even when prepared from polymers that do
However, other readily available species and strains may also
induce tumors when implanted in the form of a film. The
be acceptable for the performance of these studies. Other rat
impact of physical form and surface properties on tumorigen-
species which have been recommended include Sprague-
esis must be carefully considered, in making decisions about
Dawley, Long-Evans, and Wistar. Some investigators have
the physical form of the implants (5, 6, 7, 8, 9, 10).
recommended the use of Long-Evans or Wistar Rats because of
6.5 Researchers have found that the aspect ratio (length/
the difficulty of achieving a two-year lifespan for Fischer and
diameter) of fiber materials may play a role in the tumorigen-
Sprague-Dawley rats.
esis of a particular material (11, 12). When new fibrous
5.2 The currently accepted level of testing in a particular
materials are being tested, the actual fiber length to be
site of implantation or medical specialty should be carefully
anticipated in practice should be studied. If fragmentation can
researched and regulatory requirements determined before a
be anticipated or is a worse case possibility, an attempt should
study design is finalized to assure acceptability of the final
be made to document a clinically relevant fiber length.
results.
6.6 The material to be tested should originate from
5.3 The appropriate choice of male or female animals or a
sample(s) representative of all processing including surface
combination should be carefully considered in light of the
finishing, passivation, and sterilization or other final processing
particular material and application being investigated. If the
that will occur to a finished device.
device will ultimately be used only in the male or female, only
6.7 Dosage:
one sex may need to be evaluated. Otherwise, both sexes
6.7.1 In most materials, the ratio between the surface area of
should be used.
the implant and the body weight of the animal or person will
5.4 The decision to use other species for study should be
have an effect on the amount of extractable substances (if any)
carefully documented in terms of a clear need. The use of
which leach out of the material. The total weight or volume of
species which have not previously been used may reduce the
material used in each animal should be in excess of the
amount of comparative data available on control animals.
anticipated dosages to be seen in clinical practice when
Typical tumor rates for hamsters, rats, and mice have been
calculated based upon the ratio of surface area of sample to
tabulated and are available in Refs. (1, 2, 3).
body weight of the animal. Consideration should be given to
6. Selection of Size and Form of Implant using the maximum implantable dose as the dosage or as one
of multiple dosage levels. For the special case of degradable
6.1 Tumorigenicity bioassays have traditionally been per-
materials, the sample size should be calculated based on the
formed using chemical substances as the challenge. The
ratio of sample weight to animal body weight.
evaluation of implant materials requires that solid material be
6.7.2 Whenever possible, more than one exposure level
implanted in some form. It is important to realize that the
should be considered to evaluate a dose-response effect.
7. Choice of Control
The boldface numbers in parentheses refer to the list of references at the end of
this guide. 7.1 Control groups for this type of study will usually consist
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
F 1439
of identical animals that have not received an implant of the detection of effects at the p < 0.05 level of significance. If
test material but have been subjected to the remainder of the attrition is occurring due to reasons which cannot be attributed
surgical procedures. Additional groups such as housing (ani- to the test articles or spontaneous tumor formation, other
mals which receive no treatment but are housed with the test factors should be considered such as environmental and food
animals) and reference control groups may be included in the and water problems. This type of attrition can adversely affect
study design. the validity of a study and the investigator should be cognizant
7.2 The investigator should consider a negative control of the importance of prompt investigation of attrition in animal
group in addition to the sham or untreated controls. These numbers.
animals would receive an implant or treatment identical to the
10. Housing and Postoperative Care
test animals but the implant would be manufactured from a
10.1 The animals shall be housed and care provided in
selected negative reference material. This group would then
accordance with the Guide for the Care and Use of Laboratory
serve to isolate any results due to the implant trauma or
Animals (13) or other appropriate guidelines.
mechanically induced changes.
10.2 In addition to the requirements for humane treatment
8. Size of Test Groups
of animals in 10.1, the facilities and environment used, as well
8.1 The test group and the control group should each as any postoperative drug therapies or other treatments of the
contain enough animals which will be scheduled to survive to animals, must be carefully considered to prevent unexpected
the end of the study to allow statistically valid conclusions to effects on the results of the study. The recommendation in 7.1
be drawn from the study. If both male and female animals are that a housing control be considered is related to the possibility
being used, each group should contain an equal number of that environmental factors could provide unexpected changes
animals of each sex. The National Toxicology Program in study results if adequate care is not taken to eliminate the
requires 60 animals/sex/group for chemical studies with ten possibility.
animals being sacrificed earlier than two years. Other interna-
5 11. Evaluation of Results
tional organizations recommend 50 animals/sex/group. The
11.1 The test and control animals should be examined on a
investigator should ascertain that the number of animals in
daily basis and any remarkable observations noted. This
each group is adequate for statistical and regulatory purposes
examination should include noticeable changes in eating hab-
before proceeding. In order to ensure valid data analysis, the
animals should be randomly assigned to control and experi- its, alertness, or obvious loss in body weight and palpation at
least weekly for detectable masses. A complete record should
mental groups. Considerations specific to the particular implant
application or medical specialty may
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