ASTM F1439-03(2008)
(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
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.
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 F 748. 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 F 748 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 E 1262, E 1263, E 1280, and E 2186, and Practices E 1397 and E 1398. 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 2008)
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
Assay
1.3 Whenever possible, it is recommended that a battery of
E2186 Guide for Determining DNA Single-Strand Damage
genotoxicity procedures be initiated and proposed as an alter-
in Eukaryotic Cells Using the Comet Assay
native to an in-vivo tumorigenicity bioassay. Genotoxicity
F748 PracticeforSelectingGenericBiologicalTestMethods
assays may also be considered as initial screening procedures
for Materials and Devices
due to the sensitivity of the assays, the significant reduction in
2.2 Other Documents:
time to gain valuable data, and the desire to reduce the use of
animals for testing. Genotoxicity assays that may be consid- National Toxicology Program General Statement of Work
fortheConductofToxicityandCarcinogenicityStudiesin
ered are outlined in Guides E1262, E1263, E1280, and E2186,
and Practices E1397 and E1398. Additionally, other genotox- Laboratory Animals
OECD Guidelines for Testing of Chemicals: Guideline 451,
icity testing which might be considered (but which do not yet
Carcinogenicity Studies
have ASTM test methods) include Salmonella/Mammalian-
OECD Guidelines for Testing of Chemicals: Guideline 453,
Microsomal Plate Incorporation Mutagenicity Assay, In Vivo
Combined Chronic Toxicity/Carcinogenicity Studies
Cytogenetics Bone Marrow Chromosomal Damage Assay,
Good Laboratory Practice for Nonclinical Laboratory Stud-
BALB/3T3 Morphological Transformation of Mouse Embryo
ies
Cells, and the Mouse Micronucleus Assay. The investigator is
advisedtoconsidercarefullytheappropriatenessofaparticular
3. Terminology
method for his application after a review of the published
3.1 Definitions of Terms Specific to This Standard:
literature.
1.4 This standard does not purport to address all of the
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
safety concerns, if any, associated with its use. It is the
contact ASTM Customer Service at service@astm.org. For Annual Book ofASTM
responsibility of the user of this standard to establish appro-
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Available from National Institute of Environmental Health Sciences, Research
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Triangle Park, NC, August 1988.
Surgical Materials and Devices and is the direct responsibility of Subcommittee Available from Organization for Economic Cooperation and Development, 200
F04.16 on Biocompatibility Test Methods. L St., NW, Suite 650, Washington, DC 20036–4922.
Current edition approved Aug. 1, 2008. Published August 2008. Originally Available from 21 CFR, Part 58, U.S. Government Printing Office, Superin-
approved in 1992. Last previous edition approved in 2003 as F1439 – 03. DOI: tendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC
10.1520/F1439-03R08. 20401.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1439 − 03 (2008)
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
amount of comparative data available on control animals.
3.1.2 maximum implantable dose—the maximum weight or
Typical tumor rates for hamsters, rats, and mice have been
volume of the test article which can be reasonably implanted
tabulated and are available in Refs. (1, 2, 3).
into the test site taking into account the gross distention of
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
6.1 Tumorigenicity bioassays have traditionally been per-
induces alterations in the genetic code of the cell.
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
be considered if different types of tissue will be contacted. (For
appropriate for all applications or types of implant materials.
These recommendations should be utilized by experienced instance, materials that may be in contact with bone or
implanted into internal organ tissue might be tested in both
testing personnel in conjunction with other pertinent informa-
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
response that might be seen if the material itself were to be
5.1 These types of bioassays for chemical substances have
implanted. In general, an extract should not be used as a
traditionally been performed in mice or rats, or both, because
substitute for the actual material of interest.
of their small size, relative cost factors, and lifespan. For the
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
finishing,passivation,andsterilizationorotherfinalprocessing
combination should be carefully considered in light of the
that will occur to a finished device.
particular material and application being investigated. If the
device will ultimately be used only in the male or female, only
one sex may need to be evaluated. Otherwise, both sexes
The boldface numbers in parentheses refer to the list of references at the end of
should be used. this guide.
F1439 − 03 (2008)
6.7 Dosage: of animals per group, that fact should be documented and the
study design should proceed accordingly.
6.7.1 Inmostmaterials,theratiobetweenthesurfaceareaof
the implant and the body weight of the animal or person will
have an effect on the amount of extractable substances (if any) 9. Duration of Study
which leach out of the material. The total weight or volume of
9.1 Recommended durations for evaluation of tumorigenic-
material used in each animal should be in excess of the
ity in rats is two years.
anticipated dosages to be seen in clinical practice when
9.2 Depending upon the material being evaluated, the early
calculated based upon the ratio of surface area of sample to
results may suggest that the study can be terminated earlier
body weight of the animal. Consideration should be given to
than two years without compromising the validity of the study.
using the maximum implantable dose as the dosage or as one
Examples might include studies in which a significantly
of multiple dosage levels. For the special case of degradable
increased rate of tumor formation or toxicity is being seen in
materials, the sample size should be calculated based on the
the test animals or in one or more dosage groups.
ratio of sample weight to animal body weight.
6.7.2 Whenever possible, more than one exposure level
9.3 Attheterminationofthestudy,amajorityoftheanimals
should be considered to evaluate a dose-response effect.
in each group should have survived for euthanasia or been
terminated early for study-related reasons such as increased
7. Choice of Control
tumor incidence, spontaneous tumors, or toxicity of the test
article. It is expected that a minimum of 50 % of the animals
7.1 Control groups for this type of study will usually consist
per sex and per group should survive until final study termi-
of identical animals that have not received an implant of the
nation barring the above reasons. Moreover, the number of
test material but have been subjected to the remainder of the
survivors or study-related terminations should be sufficient for
surgical procedures. Additional groups such as housing (ani-
detection of effects at the p < 0.05 level of significance. If
mals which receive no treatment but are housed with the test
attrition is occurring due to reasons which cannot be attributed
animals) and reference control groups may be included in the
to the test articles or spontaneous tumor formation, other
study design.
factors should be considered such as environmental and food
7.2 The investigator should consider a negative control
and water problems. This type of attrition can adversely affect
group in addition to the sham or untreated controls. These
the validity of a study and the investigator should be cognizant
animals would receive an implant or treatment identical to the
of the importance of prompt investigation of attrition in animal
test animals but the implant would be manufactured from a
numbers.
selected negative reference material. This group would then
serve to isolate any results due to the implant trauma or
10. Housing and Postoperative Care
mechanically induced changes.
10.1 The animals shall be housed and care provided in
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 treatments of the
be drawn from the study. If both male and female animals are
animals, must be carefully considered to prevent unexpected
being used, each group should contain an equal number of
effec
...
This document is not anASTM standard and is intended only to provide the user of anASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation:F1439–02 Designation: F 1439 – 03 (Reapproved 2008)
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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This guide is intended to assist the biomaterials testing laboratory in the conduct and evaluation of tumorigenicity tests to
evaluatethepotentialfornewmaterialstoevokeaneoplasticresponse.Theprocedureisgenerallyreservedonlyforthosematerials
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 F 748. 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 F 748 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 E 1262, E 1263, E 1280, and E 2186, and Practices
E 1397 and E 1398. 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
MicronucleusAssay.Theinvestigatorisadvisedtoconsidercarefullytheappropriatenessofaparticularmethodforhisapplication
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.
2. Referenced Documents
2.1 ASTM Standards:
E 1262 Guide for the Performance of the Chinese Hamster Ovary Cell/Hypoxanthine Guanine Phosphoribosyl Transferase
Gene Mutation Assay
E 1263 Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes
E 1280 Guide for Performing the Mouse Lymphoma Assay for Mammalian Cell Mutagenicity
E 1397Practices for the In-Vitro Rat Hepatocyte DNARepairAssay Practice for In Vitro Rat Hepatocyte DNARepairAssay
E 1398Practices for the In-Vivo Rat Hepatocyte DNARepairAssay Practice for In Vivo Rat Hepatocyte DNARepairAssay
E 2186 Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
F 748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
2.2 Other Documents:
National Toxicology Program General Statement of Work for the Conduct of Toxicity and Carcinogenicity Studies in
Laboratory Animals
OECD Guidelines for Testing of Chemicals: Guideline 451, Carcinogenicity Studies
This guide is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.16
on Biocompatibility Test Methods.
Current edition approved Nov. 10, 2002. Published January 2003. Originally approved in 1992. Last previous edition approved in 1996 as F1439–92 (1996).
Current edition approved Aug. 1, 2008. Published August 2008. Originally approved in 1992. Last previous edition approved in 2003 as F 1439 – 03.
Annual Book of ASTM Standards, Vol 11.05.
ForreferencedASTMstandards,visittheASTMwebsite,www.astm.org,orcontactASTMCustomerServiceatservice@astm.org.ForAnnualBookofASTMStandards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Annual Book of ASTM Standards, Vol 13.01.
Available from National Institute of Environmental Health Sciences, Research Triangle Park, NC, August 1988.
Available from National Institute of Environmental Health Sciences, Research Triangle Park, NC, August 1988.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F 1439 – 03 (2008)
OECD Guidelines for Testing of Chemicals:Guideline453,CombinedChronicToxicity/CarcinogenicityStudies Guideline453,
Combined Chronic Toxicity/Carcinogenicity Studies
Good Laboratory Practice for Nonclinical Laboratory Studies
3. Terminology
3.1 Definitions of Terms Specific to this Standard:
3.1.1 carcinogenic—a substance is considered to be carcinogenic if it can be shown to be causally related to an increased
incidence of malignant neoplastic formation.
3.1.2 maximum implantable dose—the maximum weight or volume of the test article which can be reasonably implanted into
the test site taking into account the gross distention of tissue which can occur and its possible effects on test results.
3.1.3 mutagenic—a substance is said to be mutagenic if it induces alterations in the genetic code of the cell.
3.1.4 tumorigenic—a substance is said to be tumorigenic if it can be shown to be causally related to an increased incidence of
neoplastic formation whether malignant or benign.
4. 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.
5. Choice of Animal Model
5.1 Thesetypesofbioassaysforchemicalsubstanceshavetraditionallybeenperformedinmiceorrats,orboth,becauseoftheir
small size, relative cost factors, and lifespan. For the testing of biomaterials, mice are not recommended because the small animal
size is not conducive to the placement of solid implants.The investigator should seriously consider the use of one of the traditional
models in order to draw upon the extensive information available about typical tumor formation rates and sites in control animals.
The NationalToxicology Program recommends the use of Fischer 344 (F344/N) rats. However, other readily available species and
strains may also be acceptable for the performance of these studies. Other rat species which have been recommended include
Sprague-Dawley, Long-Evans, and Wistar. Some investigators have recommended the use of Long-Evans or Wistar Rats because
of the difficulty of achieving a two-year lifespan for Fischer and Sprague-Dawley rats.
5.2 Thecurrentlyacceptedleveloftestinginaparticularsiteofimplantationormedicalspecialtyshouldbecarefullyresearched
and regulatory requirements determined before a study design is finalized to ensure acceptability of the final results.
5.3 The appropriate choice of male or female animals or a combination should be carefully considered in light of the particular
materialandapplicationbeinginvestigated.Ifthedevicewillultimatelybeusedonlyinthemaleorfemale,onlyonesexmayneed
to be evaluated. Otherwise, both sexes should be used.
5.4 The decision to use other species for study should be carefully documented in terms of a clear need. The use of species
whichhavenotpreviouslybeenusedmayreducetheamountofcomparativedataavailableoncontrolanimals.Typicaltumorrates
for hamsters, rats, and mice have been tabulated and are available in Refs. (1, 2, 3).
6. Selection of Size and Form of Implant
6.1 Tumorigenicity bioassays have traditionally been performed using chemical substances as the challenge. The evaluation of
implant materials requires that solid material be implanted in some form. It is important to realize that the down-sized implants
necessaryforuseinanimalswillhaveagreatersurfaceareatovolumeratio,andthisdifferencemustbeconsideredinexperimental
design.
6.2 It may be important to determine the site of administration of the test material that is most appropriate to the end use before
determining implant size. The site of implantation should be the paravertebral muscle unless the size of the implant causes this
site to be unacceptable.Alternatively, the site of implantation should mimic the anticipated end use, if possible. Where a specific
material may be utilized in more than one type of device, multiple sites of administration should be considered if different types
of tissue will be contacted. (For instance, materials that may be in contact with bone or implanted into internal organ tissue might
be tested in both tissues.)
6.3 It should be recognized that the response of the test animal to an extract of a material may not fully represent the response
that might be seen if the material itself were to be implanted. In general, an extract should not be used as a substitute for the actual
material of interest.
Available from Organization for Economic Cooperation and Development, 200 L St., NW, Suite 650, Washington, DC 20036–4922.
Available from Organization for Economic Cooperation and Development, 200 L St., NW, Suite 650, Washington, DC 20036–4922.
Available from 21 CFR, Part 58, U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
Available from 21 CFR, Part 58, U.S. Government Printing Office, Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401.
The boldface numbers in parentheses refer to the list of references at the end of this guide.
F 1439 – 03 (2008)
6.4 The physical form of the test material should be representative of that intended for use in human patients and should
consider potential material debris, if appropriate. The investigator should be aware that tests have shown (4) that powdered
polymeric materials may not elicit a tumorigenic response subcutaneously even when prepared from polymers that do induce
tumors when implanted in the form of a film. The impact of physical form and surface properties on tumorigenesis must be
carefully considered, in making decisions about the physical form of the implants (5, 6, 7, 8, 9, 10) .
6.5 Researchers have found that the aspect ratio (length/diameter) of fiber materials may play a role in the tumorigenesis of a
particular material(11,12).When new fibrous materials are being tested, the actual fiber length to be anticipated in practice should
be studied. If fragmentation can be anticipated or is a worse case possibility, an attempt should be made to document a clinically
relevant fiber length.
6.6 The material to be tested should originate from sample(s) representative of all processing including surface finishing,
passivation, and sterilization or other final processing that will occur to a finished device.
6.7 Dosage:
6.7.1 In most materials, the ratio between the surface area of the implant and the body weight of the animal or person will have
an effect on the amount of extractable substances (if any) which leach out of the material. The total weight or volume of material
used in each animal should be in excess of the anticipated dosages to be seen in clinical practice when calculated based upon the
ratio of surface area of sample to body weight of the animal. Consideration should be given to using the maximum implantable
dose as the dosage or as one of multiple dosage levels. For the special case of degradable materials, the sample size should be
calculated based on the ratio of sample weight to animal body weight.
6.7.2 Whenever possible, more than one exposure level should be considered to evaluate a dose-response effect.
7. Choice of Control
7.1 Control groups for this type of study will usually consist of identical animals that have not received an implant of the test
material but have been subjected to the remainder of the surgical procedures. Additional groups such as housing (animals which
receive no treatment but are housed with the test animals) and reference control groups may be included in the study design.
7.2 Theinvestigatorshouldconsideranegativecontrolgroupinadditiontotheshamoruntreatedcontrols.Theseanimalswould
receive an implant or treatment identical to the test animals but the implant would be manufactured from a selected negative
reference material. This group would then serve to isolate any results due to the implant trauma or mechanically induced changes.
8. Size of Test Groups
8.1 The test group and the control group should each contain enough animals which will be scheduled to survive to the end of
the study to allow statistically valid conclusions to be drawn from the study. If both male and female animals are being used, each
requires 60 animals/sex/group
group should contain an equal number of animals of each sex. The National Toxicology Program
for chemical studies with ten animals being sacrificed earlier than two years. Other international organizations recommend 50
animals/sex/group. The investigator should ascertain that the number of animals in each group is adequate for statistical and
regulatory purposes before proceeding. In order to ensure valid data analysis, the animals should be randomly assigned to control
and experimental groups. Considerations specific to the particular implant application or medical specialty may mandate a greater
number of animals in each group. Additional animals in interim sacrifice groups or satellite groups may be added.
8.2 The number of test animals in each group shall be determined based upon a sound statistical analysis of the scientific
questions to be addressed by the study. This analysis should take into account predicted survival rates (
...
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