United States Prevention, Pesticides EPA712-C-96-281
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Biochemicals Test
Guidelines
OPPTS 880.3800
mmune Response
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 for disks or paper copies.
This guideline is also available electronically in ASCII and PDF (portable
document format) from the EPA Public Access Gopher (gopher.epa.gov)
under the heading "Environmental Test Methods and Guidelines."
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OPPTS 880.3800 Immune response.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 152-24.
(b) Immunotoxicity studies with biochemical pest control agents
(BPCAs): tier II—When required. Data on alterations of immune re-
sponses (tier II) are required by 40 CFR 158.165 to support the registration
of each manufacturing-use and each end-use product when significant
immunotoxic effects are observed in test animals treated in any of the
tier I immunotoxicity studies (OPPTS 880.3550), or when the
immunotoxicity data from the tier I studies cannot be definitively inter-
preted, or when there is available data from other sources which indicate
that the test substance, or structurally-related substances (including
metabolites and degradation products) are immunotoxic.
(c) Test standards—(1) Principles of the test methods. The tests
and methods are designed to:
(i) Provide information on the time-course of recovery from each sig-
nificant adverse immunotoxic effect observed in tier I studies.
(ii) Indicate whether the observed effects may result in impaired host
resistance to infectious microbial agents and/or to tumor cell challenge.
(iii) To provide additional information essential for a full evaluation
of potential risks associated with the immunotoxicityof a test substance.
(2) Test selection, (i) Selection of the appropriate tests to be done
in tier II studies will depend on:
(A) The particular test or tests in tier I in which significant
immunotoxic effects were observed.
(B) The availability of data from other sources which indicate that
the test substance, or structurally-related substances, are immunotoxic.
(C) Whether data from initial testing in tier II indicate that expanded
testing is required, using additional tests in this tier.
(ii) Consultation with appropriate representatives of the Agency is
suggested to determine which tests are to be performed.
(d) Immunotoxicity studies—(1) Recovery from adverse
immunotoxic effects. For each study in tier I (OPPTS 880.3550) where
significant immunotoxic effects were observed (including histopathological
effects), the satellite group of treated test animals (OPPTS 880.3550(c)(3))
is to be used to evaluate the time-course for recovery from the effects
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after withdrawal of the test substance. The tests should be conducted in
accordance with the protocols set forth for each test in OPPTS 880.3550,
with the following modifications:
(i) At least five treated animals in the satellite group and five un-
treated control animals are to be evaluated at 7, 14, and 28 days after
the withdrawal of the test substance.
(ii) Only those tests in OPPTS 880.3550 in which significant
immunotoxic effects were observed need be repeated with the satellite
group of test animals.
(iii) If a satellite group of test animals, or control animals for the
satellite group, were not included as suggested in the tier I studies, or
if these animals were insufficient in number for proper evaluation of recov-
ery from any adverse immunotoxic effects observed in the tier I studies,
then the necessary tests should be initiated as described in OPPTS
880.3550, and completed as described above with the appropriate numbers
of test and control animals.
(iv) Studies in tier I may be required, in which both sexes of several
strains of test animals are exposed to the BPCA at several dose levels
for longer time periods (e.g., 90 days).
(2) Altered host resistance after challenge with infectious agents
or with tumor cells, (i) The selection of an appropriate host resistance
model (or models) will depend largely on the particular immunotoxic ef-
fects observed from testing at the tier I level (OPPTS 880.3550) and, on
whether the model system has been established as providing reproducible
data for detecting altered host resistance after exposure to environmental
chemicals.
(ii) Host resistance models that may be appropriate for use when alter-
ations of cell-mediated immune responses are observed in tier I tests in-
clude challenge with Listeria monocytogenes or challenge with transplant-
able syngeneic (or semi-syngeneic) tumor cells, including PYB6
fibrosarcoma or B16F10 melanoma tumor of C57BL/6 mice; or challenge
with Herpes simplex virus type 1 or type 2 (HSV-1, HSV-2).
(iii) Host resistance models that may be appropriate for use when
alterations of humoral-mediated immune responses are observed in tier I
tests include challenge with Streptococcus sp. (e.g., Streptococcus
pyogenes) or challenge with HSV-1 or HSV-2.
(iv) Other models, not mentioned above, involving challenge with
other infectious agents or tumor cells, also may prove useful in evaluating
host resistance to BPCAs.
(v) It may be considered appropriate to include host resistance models
as substitutes for certain immunotoxicity tests recommended in tier I
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(OPPTS 880.3550). However, documentation must be provided to show
that the immune system parameters measured by the selected host resist-
ance models are equivalent to those evaluated in the tests suggested in
tier I.
(vi) General test standards. (A) The resistance of host animals to chal-
lenge by infectious agents or tumor cells usually will involve exposing
young adult rodents for 30 days to the highest dose of BPCA that did
not lead to overt toxicity in the test animals.
(B) Reporting on parameters, appropriate for each host resistance
model system, other than the endpoint of mortality, should be considered
so that proper interpretation can be made of any observed changes in host
resistance.
(C) Appropriate positive control groups of test animals, dosed with
a known immunosuppressive chemical, should be included with each host
resistance system used.
(D) In addition to providing all relevant data appropriate for each
host resistance system used, protocols should be sufficiently well-de-
scribed, and justification/ reasoning should be provided for any modifica-
tions in protocols of standard techniques.
(E) Current knowledge on the immune system components involved
in host resistance to the infectious agent or tumor cell should be included
in the report.
(F) In-depth host resistance studies may be required, in which several
strains and both sexes of host animal (weanling to aged) are exposed to
the BPCA at several dose levels for longer time periods (e.g., 90 days).
(3) Other immunotoxicological studies, (i) Additional tests may be
required if considered necessary for a full evaluation of potential risks
associated with the immunotoxicity of a BPCA. These include, but are
not necessarily limited to, available tests that are designed to evaluate ef-
fects of a substance on:
(A) Lymphoid organs and tissues (using enzyme and immune
histochemistry).
(B) Serum complement.
(C) Antibody response to T-independent antigens.
(D) Enumeration of subpopulations of T- and B- lymphocytes.
(E) Granulocyte function.
(F) Bone marrow function.
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(G) Lymphokines.
(H) Plaque-forming cell response to T-independent antigens.
(I) Mitog stimulation of lymphocyte blastogenesis.
(J) In vivo popliteal lymph node enlargement after injection of
allogeneic lymphocytes.
(K) Hormones.
(L) Immune system development.
(M) Macrophage development, activation and function.
(N) Induction of autoimmunity.
(ii) Certain additional tests may be required if evidence is obtained
which indicates that the test substance is immunogenic.
(iii) When required. Tests to measure these and other parameters of
the immune system may be required when indicated by:
(A) The extent and/or nature of the immunotoxic effects observed
in the tier I (OPPTS 880.3550) tests.
(B) The nature of immunotoxic effects observed in the host resistance
challenge models.
(C) The inability to definitively interpret the data from tier I studies
or from the host resistance challenge models;
(D) The necessity to conform, by using alternate tests, the results from
tier I studies.
(E) The availability of data from other sources which indicate that
the test substance, or closely related substances (including metabolites and
degradation products) are immunotoxic.
(e) References. The following references should be consulted for ad-
ditional background material on this test guideline. The following are pub-
lications that either provide useful protocols for the design of
immunotoxicity studies, or contain citations for useful protocols.
(1) Cunningham, A.J. A method of increased sensitivity for detecting
single antibody-forming cells. Nature 207:1106-1107 (1965).
(2) Cunningham, A.J., and A. Szenberg. Further improvements in the
plaque technique for detecting single antibody-forming cells. Immunology
14:599-601 (1968).
(3) Daugherty, M.L. Immunotoxicology; in Scientific Rationale for
the Selection of Toxicity Testing Methods. II. Teratology,
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Immunotoxicology, and Inhalation Toxicology. M.G. Ryon and D.S.
Sawhney, eds. EPA-560/6-84-004 (1985).
(4) Dean, J.H. et al. Amos (eds). Immunotoxicology and
Immunopharmacology. Raven Press, NY (1985).
(5) Dean, J.H. et al. Toxic responses of the immune system. Pp. 245-
285; in Casarett and DouU's Toxicology: The Basic Science of Poisons.
Third edition. J. Doull, C.D. Klaassen, and M.O. Amdur (eds). MacMillan,
NY (1987).
(6) Exon, J.H. et al. Effect of lead, polychlorinated biphenyls and
cyclophosphamide on rat natural killer cells, interleukin 2 and antibody
synthesis. Fundamental and Applied Toxicology 5:158-164 (1985).
(7) Jerne, N.K., and A.A. Nordin. Plaque formation in agar by single
antibody-producing cells. Science 140:405-406 (1963).
(8) Klein, J. Immunology. The Science of Self-Nonself Recognition.
John Wiley and Sons, NY (1982).
(9) Luster, M.I. et al. Evaluation of immune functions in toxicology.
Pp. 561-586 in Principles and Methods in Toxicology. A.W. Hayes (ed).
Raven Press, NY (1982).
(10) Luster, M.I. et al. Development of a testing battery to assess
chemical-induced immunotoxicity: National Toxicology Program's guide-
lines for immunotoxicity evaluation in mice. Fundamental and Applied
Toxicology 10:2-19 (1988).
(11) Mishell, R. and R. Dutton. Immunization of dissociated mouse
spleen cell cultures from normal mice. Journal of Experimental Medicine
126:423-442 (1967).
(12) Norbury, K.C. Immunotoxicological evaluation: an overview.
Journal of the American College of Toxicology 4:279-290 (1985).
(13) Proceedings. (1978) Inadvertant Modification of the Immune Re-
sponse. The Effects of Foods, Drugs, and Environmental Contaminants.
I.M. Asher, ed. Fourth FDA Science Symposium. Office of Health Affairs,
FDA.
(14) Proceedings. (1982) Target Organ Toxicity: Immune System. En-
vironmental and Health Perspectives 43. Symposium held at Arlington,
VA, October 20-21, 1980. Sponsored by Society of Toxicology and
NIEHS.
(15) Proceedings. (1983) NIH Immunotoxicology Workshop. Sympo-
sium held at Research Triangle Park, NC, October 17-18, 1983. Sponsored
by NIH and NIEHS.
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(16) Proceedings. (1985) International Seminar on the Immunological
System as a Target for Toxic Damage. Symposium held at Luxembourg,
Belgium, November 6-9, 1984. Sponsored by UNEP, ILO, WHO and
COEC.
(17) Reynolds, C.W., and R.B. Herberman. In vitro augmentation of
rat natural killer (NK) cell activity. Journal of Immunology 126:1581-1585
(1984)
(18) Riccardi, C. et al. Rapid in vivo assay of mouse natural killer
cell activity. Journal of the National Cancer Institute. 63:1041-1045.
(1979).
(19) Riccardi, C. et al. Role of natural killer cells in rapid in vivo
clearance of radiolabelled tumor cells. Pp. 1121-1139. In Natural Cell Me-
diated Immunity Against Tumors. R.B. Herberman (ed.) Academic Press,
NY (1980).
(20) Rose, N.R., and H. Friedman (eds.). Manual of Clinical Immu-
nology. ASM, Washington DC (1980).
(21) Stites, D.P. et al. Basic and Clinical Immunology. Lange Medical
Publications, Los Altos, CA (1984).
(22) Temple, L. et al. Comparison of ELISA and plaque-forming cell
assays for measuring the humoral immune response to SRBC in rats and
mice treated with benzo[a]pyrene or cyclophosphamide. Fundamental and
Applied Toxicology 21:412-41 (1993).
(23) Tripathy, S.P., and G.B. Mackaness. The effect of cytotoxic
agents on the primary immune response to Listeria monocytogenes. Jour-
nal of Experimental Medicine 130:1-16 (1969).
(24) Van Furth, R. et al. In vitro determination of phagocytosis and
intracellular killing by polymorphonuclear and mononuclear phagocytes.
Pp. 32.1-32.19 In: TSHandbook of Experimental Immunology D.M. Weir
(ed.) Blackwell, Oxford (1978).
(25) Vos, J.G. et al. Ovalbumin immunity in the rat: Simultaneous
testing of IgM, IgG, and IgE response measured by delayed-type
hypersensitivity. Scandinavian Journal of Immunology 12:289-295 (1980).
(26) Vos, J.G. et al. Use of the Enzyme-linked immunosorbent assay
(ELISA) in immunotoxicity testing. Environmental and Health Perspec-
tives 43:115-121 (1982)
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