United States       Prevention, Pesticides     EPA712-C-96-331
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA   Microbial Pesticide
          Test Guidelines
          OPPTS 885.4150
          Wild Mammal Testing,

     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 885.4150  Wild mammal testing, Tier I.
     (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 154A-18. The toxicity
and pathogenicity data required for  evaluating hazard to humans and do-
mestic animals  are normally adequate to indicate potential hazard to wild
mammals. Under  certain conditions, however, these data are not sufficient
to assess the potential hazard to wild mammals likely to be exposed to
a microbial pest control agent (MPCA).  An example of one circumstance
when such additional testing may be required is the situation in  which
data indicate that  there is considerable variation in sensitivity of different
mammalian species to the effects of an MPCA agent, and there is evidence
that  wild mammals will be  heavily exposed  to an MPCA. See  40 CFR
158.50 and 158.740(e) to determine whether these data must be submitted.

     (b) Test standards. Data must be derived from tests that satisfy the
general test standards in OPPTS 885.0001 and the following:

     (1) Test substance. The  actual form of the material to be regarded
as the test substance is described in  section OPPTS 885.0001. In addition,
any substances used to enhance virulence or toxicity should be tested along
with the test substance.

     (2) Species. Testing shall be performed on a mammalian species rep-
resentative or indicative of those found in areas likely to be affected by
the proposed use patterns. Test animals may be reared in pens or captured
in the wild, and must  be phenotypically indistinguishable from wild mam-
mals. Endangered or threatened animals shall not be used.

     (3) Controls, (i) A negative control group is required.

     (ii) A concurrent control group is required and shall be treated, when
possible, with the pure active ingredient that has been inactivated in such
a way as to preserve cellular integrity.

     (4) Route  of exposure. The test material should be administered by
gavage (acute oral dose) or by intranasal instillation. The method of dosing
should reflect the expected exposure route  and shall be determined after
consultation with the Agency.

     (5) Maximum hazard dosage level. The  standards for maximum haz-
ard dosage level,  determination of an  LD50 or ID50, and duration  of test
that are found in the avian oral pathogenicity/toxicity test OPPTS 885.4050
and the avian inhalation pathogenicity test OPPTS 885.4100.

     (c) Reporting and evaluation of data. In addition to the information
specified in OPPTS 885.0001, test reports shall contain the same informa-

tion required for the avian oral pathogenicity/toxicity test OPPTS 885.4050
and the avian  inhalation pathogenicity test OPPTS 885.4100, adapted ap-
propriately for mammalian test procedures.

     (d) Tier progression. (1) If any toxic or pathogenic effects on mam-
malian species are observed at the maximum hazard dosage level in this
study,  testing  at Tier  II  (OPPTS  885.5000,  885.5200, 885.5300,  and
885.5400—environmental expression  testing) is required as specified in
40  CFR 158.740(e).  In  some cases, a subchronic test may serve to better
understanding  of the the effects observed at the Tier I level and alleviate
the need for Tier II testing.

     (2) If toxic or pathogenic effects  are not observed  in this study, addi-
tional testing at higher  tiers ordinarily is not required. The Agency may
require additional testing, however, if it determines that there is  a potential
risk to mammals despite negative Tier I results.

     (e) References. The following references are provided for use in the
development of test protocols for conducting  wild mammal toxicity and
pathogenicity tests with microbial pest control agents:

     (1) Barnes, R.W. et  al. Long-term feeding and other toxicity/patho-
genicity studies on rats using  a commercial preparation of the nuclear-
polyhedrosis virus  of Heliothis zea.  Journal of Invertebrate  Pathology

     (2) Fisher, R. and L.  Rosner. Toxicology of the microbial insecticide,
Thuricide. Agricultural and Food Chemistry 7:686-688  (1959).

     (3) Ignoffo, C.M. and A.M. Heimpel.  The  nuclear polyhedrosis virus
of Heliothis zea (Boddie)  and Heliothis virescens (Fabricius) Part V. Tox-
icity-pathogenicity of virus to  white  mice and guinnea  pigs. Journal of
Invertebrate Pathology 7:329-340 (1965).

     (4)  Ignoffo,  C.M.  Intraperitoneal  injection of  white  mice  with
nucleopolyhedrosis virus of the beet armyworm, Spodoptera exigua. Jour-
nal of Invertebrate Pathology 17:453-454 (1971).

     (5) Ignoffo, C.M. Effects of entomopathogens on vertebrates.  Annals
N.Y. Academy of Science 217:141-164 (1973).

     (6) Ignoffo, C.M.  et al. Lack  of susceptibility of mice and  rats to
the mosquito nematode,  Reesimermis nielseni, Tsai and Grundmann. Mos-
quito News 34:425-428 (1974).

     (7) Ignoffo,  C.M. et al. An evaluation of the risks  to mammals of
the use  of an  entomopathogenic fungus,  Nomuraea rileyi, as a microbial
insecticide.  In: Baculoviruses for Insect Pest Control: Safety  Consider-
ations. Selected papers from EPA/USDA Working Symposium, American
Society of Microbiologists, Washington, DC (1975).

     (8) Lamanna, C. and L. Jones.  Lethality for mice of vegetative and
spore forms of Bacillus cereus and Bacillus  cereus-like insect pathogens
injected  intraperitoneally  and  subcutaneously. Journal  of Bacteriology
85:532-535 (1963).

     (9) Lautenschlager, R.A.et al. Effect of nucleopolyhedrosis virus on
selected mammalian predators of the gypsy moth. USDA Forest Service
Research Papers, NE-377, 6p. (1977).

     (10)   Lautenschlager,  R.A.   and  J.D.  Podgwaite.  Passage   of
nucleopolyhedrosis virus by avian and mammalian predators of the gypsy
moth, Lymantria dispar. Environmental Entomology 8:210-214 (1979).

     (11) Lautenschlager, R.A.  and J.D. Podgwaite. Passage of infectious
nuclear-polyhedrosis virus through the alimentary tracts of two small mam-
mal predators of the gypsy moth, Lymantria  dispar. Environmental Ento-
mology 6:737-738 (1977).

     (12) Meinacke, C.F. et al. Toxicity-pathogenicity studies of a nuclear-
polyhedrosis virus of Heliothis zea in white mice. Journal of Invertebrate
Pathology  15:10-14 (1970).

     (13) Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds. Pp. 179-
184 In: Guidelines for Safety Testing of Baculoviruses Baculoviruses for
Insect Pest Control: Safety Considerations.  American Society for Microbi-
ology Washington, DC (1975).

     (14) Watts, D.M. et al. Experimental  infection of vertebrates of the
Pocomoke  Cypress Swamp, Maryland with Keystone and Jamestown Can-
yon viruses. American Journal of Tropical Medicine and Hygiene 28:344-
350 (1979).

     (15) Wolf,  K. Evaluation of the exposure of fish and wildlife to nu-
clear polyhedrosis and granulosis viruses. Pp. 109-111 In: Baculoviruses
for  Insect  Pest Control:  Safety Considerations.  American Society for
Microbiology, Washington, DC  (1975).