Designation: F 1905 – 98 (Reapproved 2003)

Standard Practice For

Selecting Tests for Determining the Propensity of Materials
to Cause Immunotoxicity1
This standard is issued under the fixed designation F 1905; 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 the antigen. These cells do not circulate widely in the host and
1.1 This practice covers the introduction of foreign materi- are generally located at the site of antigen deposition. The use
als into the body that may have an impact on the immune of living lymphocytes is required to test for CMI to an antigen.
system. One possible effect is that the immune system will be 3.1.3 complement—this is a complex system of circulating
depressed or certain cell types may be affected. Immunotoxic- proteins (enzymes, pro-enzymes, and co-factors) found in the
ity may be determined with blood and tissue samples from the blood. This system is usually activated by antigen-antibody
animals used in the other biocompatibility test procedures such reactions and is a reflection of humoral immunity. However it
as implantation and blood contact test protocols. It is also is apparent that other factors can activate the complement
possible to use these techniques with blood samples from system. These include large polysaccharides and various ma-
human patients in a clinical trial. Any procedures with human terials and tissues. Activation of complement can affect the
subjects should follow the appropriate rules of the local immune system, inflammation, and vascular activity with fever
institutional review board and the appropriate regulatory agen- and shock as a consequence of complement activation in the
cies. This document may serve as an annex to Practice F 748. host.
1.2 The material may affect the humoral immune response, 3.1.4 humoral immunity—some antigens stimulate the host
the cell mediated response, or both. to produce antibodies (immunoglobulins) which are specific
1.3 This standard does not purport to address all of the for the antigen and react with the antigen. Antibodies circulate
safety concerns, if any, associated with its use. It is the in the blood and tissue fluids. The antibodies produced can be
responsibility of the user of this standard to establish appro- detected using blood from the host.
priate safety and health practices and determine the applica- 3.1.5 inflammatory factors—various soluble substances
bility of regulatory limitations prior to use. may be produced by lymphocytes in response to an antigen.
This may occur in humoral immune responses or in CMI.
2. Referenced Documents These substances may influence the function of other cells and
2.1 ASTM Standards: 2 are called cytokines. Many of these act on various white cells
F 619 Practice for Extraction of Medical Plastics and are called interleukins. They are reflection of antigenic
F 748 Practice for Selecting Generic Biological Test Meth- stimulation of the host.
ods for Materials and Devices
4. Summary of Practice
3. Terminology 4.1 Immunotoxicity testing is done using specimens from
3.1 Definitions: animals being tested according to the Practice F 748 matrix for
3.1.1 antigens—these are substances that stimulate the host irritation and sensitivity, or for implantation. Blood, organs, or
to produce an immune response tissues from the animals may be used. In predicting biocom-
3.1.2 cell mediated immunity (CMI)—some antigens stimu- patibility, tests using animals are recommended even though
late the production of lymphocytes that react specifically with the material will eventually be used in humans. The use of
human material is also feasible in this testing protocol for those
laboratories having approval for such studies.
1
This practice is under the jurisdiction of ASTM Committee F04 on Medical and 4.2 Immunologic testing is done using materials or extracts
Surgical Materials and Devices and is the direct responsibility of Subcommittee according to Practice F 619. These materials or extracts may be
F04.16 on Biocompatibility Test Methods.
Current edition approved Nov. 1, 2003. Published December 2003. Originally used for in vivo tests or for the in vitro tests.
approved in 1998. Last previous edition approved in 1998 as F 1905 – 98.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [email protected]. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

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 F 1905 – 98 (2003)
5. Significance and Use labeled anti-species IgM or IgD, or with an appropriate anti CD
5.1 This practice is to be used to help assess the biocom- antibody and with the use of a cell sorter for enumeration.
patibility of materials used in medical devices. It is designed to 6.2 Effects on the cell Mediated System:
test the immunotoxicity of such materials. 6.2.1 Enumeration of Lymphocytes:
5.2 The appropriateness of the methods should be carefully 6.2.1.1 Total T Cell Counts—This should be done using
considered by the user since not all materials or applications species specific labeled anti T cell CD antigens, preferably anti
need be tested by this practice. CD2 or anti CD3 and a cell sorter.
5.3 The testing suggestions in Practice F 748 and in the 6.2.2 Enumeration of T Cell Types—This should be done
matrices of recommended tests issued by regulatory agencies using species specific labeled anti CD4 and anti CD8.
such as the FDA Immunotoxicity Testing Guide may be 6.2.3 Responses of T Cells—T cells have receptors on their
considered before proceeding with these tests. surfaces for plant lectins such as PHA, ConA and they will
5.4 Abbreviations: divide and proliferate in response. T cells are incubated with
5.4.1 BSA—Bovine Serum Albumin. PHA or ConA in tissue culture for four days. The level of
5.4.2 IgG, IgA, IgM, IgE, IgD—Immunoglobulin Class G, response can be determined by cell counts or by the uptake of
Class A, Class M, Class E, Class D. tritiated thymidine.
5.4.3 ELISA—Enzyme Linked Immunosorbent Assays 6.3 Effects on Inflammatory Factors:
5.4.4 EIA—Enzyme Immunoassays. 6.3.1 Interleukins—The incubation of lymphocytes in cul-
5.4.5 RIA—Radio Immunoassays. ture with the material may lead to the stimulation of the
5.4.6 PHA—Phytohemagglutinin. production of cytokines. The most common, and the ones that
are readily detectable and quantifiable, are the interleukins,
5.4.7 ConA—Concanavalin A.
IL-1 and IL-2 are the recommended ones.
5.4.8 IL—Interleukin (identified by numbers).
6.4 Complement—Materials, especially those in devices
5.4.9 C—Complement (components are numbered or let-
that are blood contacting, or in contact with the CNS or large
tered).
extracorporeal circuits, may affect complement which is a
5.4.10 SRBC—Sheep Red Blood Cells.
series of proteins important in inflammation and the immune
5.4.11 CD—Cluster of Differentiation (antigenic markers
response. The usual method is to determine levels of a
on cells).
component of complement. Commercially available kits are
5.4.12 CNS—Central Nervous System. recommended. It is essential to follow the instructions since
serum and plasma may not be interchangeable in these tests.
6. In Vitro Tests for Immunotoxicity The use of a known nonstimulatory biomaterial and a known
6.1 Effects on the Humoral Response—The following de- stimulant such as cobra venom factor are recommended as
terminations are important in assessing the ability of a material controls. (NB: measurable effects on the complement system
to depress the immune response. generally occur only after major alterations in the immune
6.1.1 Total Immunoglobulins—Commercially available kits system.)
are recommended for measuring immunoglobulin Class spe- 6.4.1 To determine the effect on the Classical Pathway,
cific (IgG, IgM, IgA, IgD, IgE) immunoglobulins may also be measurement of C2 is recommended or the split products of
measured and commercially available kits are recommended. C4.
NOTE 1—Changes in total immunoglobulins are seen only after major
6.4.2 To determine the effect on the Alternate Pathway,
disturbance of the immune system. measurement of factor B, Bb, or D are recommended.
6.4.3 To determine the effect on the total complement
6.1.2 Effect on Specific Immune Response: system, measurement of C3 is recommended. Measurement of
6.1.2.1 The use of BSA as the antigen is recommended in the split products of C3 or C5 is highly recommended.
animal studies. Tetanus toxoid or another approved vaccine is 6.4.4 The activity of the complement system is best deter-
recommended in human clinical trials. The immune response mined with a hemolysis assay or by assay for the terminal
in those receiving the material and the antigen and the immune complex (Sc5b-9). The standard 50 % hemolytic assay (CH 50)
response in those receiving only the antigen should be deter- is still a method of choice and is described in the cited “other
mined and compared. A standard ELISA or RIA assay for the documents”.
immune response is recommended.
6.1.2.2 The use of SRBC is also recommended in animal 7. Data Analysis and Report
studies. The assay for plaque forming cells using single cell 7.1 The data obtained should be compared to the results in
suspensions from spleens harvested four days after immuniza- control animals not receiving the material. In tests where actual
tion is a sensitive test for IgM antibody. numbers are obtained these should be reported as mean and
6.1.2.3 The use of a known adjuvant such as Complete standard deviation or standard error. In some cases, the report
Freund’s Adjuvant or Titer Max is recommended. The use of of presence or absence of response or estimation of the
physiologic saline or phosphate buffered saline as a negative magnitude of the responses will suffice.
control is recommended.
6.1.3 Effect on B Cell Numbers—This provides direct infor- 8. Keywords
mation on the effect of the material on the B cell proliferation 8.1 B cells; biocompatibility; cell mediated immunity;
and killing. This should be done with commercially available, complement; humoral immunity; immunotoxicity; T cells

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 F 1905 – 98 (2003)

APPENDIXES

(Nonmandatory Information)

X1. RATIONALE

X1.1 The primary purpose of this practice is to describe years. However, not many studies have been done with medical
methodologies to determine the propensity of materials to materials. Many investigators have developed procedures for
affect the immune response. doing immunotoxicity studies. This document is intended to
delineate the information necessary for selection of tests to be
X1.2 It is well recognized that the immune response is an done.
important defense mechanism of the host. The interaction of
materials with host tissue might alter this response and it is X1.4 The interaction of the immunological system with
important to understand what materials affect which parts of materials could lead to the production of various responses. It
the immune system. is unknown at this time whether immunotoxicity which may be
caused by the materials is unfavorable to the host. Immuno-
X1.3 The nature of the immune response and features of toxicity studies using medical materials are important so that
immunotoxicity have been an active research area for many this information can be obtained.

X2. ADDITIONAL REFERENCES

Burleson, GR, Dean JH, Munson AE., Methods in Immuno- Rose NR, Friedman H., Manual of Clinical Immunology,
toxicology, Wiley-Liss, New York, 1995. Washington DC, American Society for Microbiology, 1992.
Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Wild, D., The Immunoassay Handbook, Stockton Press,
Strober W. Current Protocols in Immunology, N.Y., John 1994.
Wiley, 1992 (appended frequently).
Price, CP, Newman, DJ., Principles and Practice of Immu-
noassay, Stockton Press, NY, NY 1991.

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