United States       Prevention, Pesticides     EPA712-C-96-281
          Environmental Protection    and Toxic Substances     February 1996
          Agency         (7101)
&EPA    Biochemicals Test
          OPPTS 880.3800
           mmune Response

     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

     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,    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."

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

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

     (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

(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

     (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

     (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.

     (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,

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,

     (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

     (15) Proceedings. (1983) NIH Immunotoxicology Workshop. Sympo-
sium held at Research Triangle Park, NC, October 17-18, 1983. Sponsored
by NIH and NIEHS.

     (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

     (17) Reynolds, C.W., and R.B. Herberman. In vitro augmentation of
rat natural killer (NK) cell activity. Journal of Immunology 126:1581-1585

     (18) Riccardi, C. et al.  Rapid in  vivo assay of mouse natural  killer
cell  activity. Journal of the National Cancer Institute. 63:1041-1045.

     (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)