United States       Prevention, Pesticides     EPA712-C-96-329
          Environmental Protection    and Toxic Substances     February 1996
          Agency         (7101)
&EPA    Microbial Pesticide
           Test Guidelines
           OPPTS 885.4050
           Avian Oral, Tier

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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 885.4050 Avian oral, Tier I.
     (a) Scopeó(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-16.

     (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 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 one  avian species,  one
insectivorous and  one herbivorous (preferably bob white quail). The use
of two species of birds with differing diets  is recommended in order to
take into account possible differences in gastrointestinal physiology. Other
species may be used but  a justification must be supplied based on in-
creased susceptibility to the MPCA or ecological considerations which pre-
clude the use of recommended species.

     (3) Age. Birds  used in this  test shall be from 14 to 24 days old at
the beginning of the test period. Within  a given test, all birds shall be
as near the same age as possible.

     (4) Controls, (i) A negative, nondosed control group  should  be per-
formed.

     (ii) An infectivity control group should be  performed and should be
treated with the MPCA inactivated in such a way as to  retain the structural
integrity of the cell.

     (iii) A control group in which the birds are dosed with sterile filtrate
from production cultures should  be performed concurrently with the test
groups.

     (5) Number of birds per dosage level. Each treatment and  control
group shall  contain at least 10 birds. When only one treatment group is
tested, at least 30 birds shall be tested at that level.

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     (6) Maximum hazard dosage level, (i) The highest oral dosage level
tested is defined by the following formula:

     maximum daily dose (units) =

           [MPCA] in TGAI x 5 mL/kg BW x weight of test bird (kg)

where

     [MPCA] = concentration of MPCA

     TGAI = technical grade of active ingredient

     BW = body weight

     (ii) When using injection routes, use 0.5 mL/kg BW for intravenous,
and 2 mL/kg BW for intraperitoneal.

     (iii) For MPCAs that produce a toxin, fractions of this dose should
be calculated for lower doses. This dose is administered daily for 5  days.
A justification shall be provided to support any reduction in the highest
dosage level.

     (7) Treatment conditions. A single group of birds may be tested
at the maximum hazard dose. If deleterious effects, due either to toxicity
or pathogenicity are observed, sequentially lower doses should be tested
as described in paragraph (b)(9) of this guideline.

     (8) Dosing regimen. Birds  will receive oral doses daily for 5  days.

     (9) Determination of an LD50 or ID50. (i) The study endpoint must
be chosen to reflect the activity of the specific microorganism under test,
i.e. if an MPCA is  expected to produce a toxin and has little or no infectiv-
ity, the appropriate endpoint would be death of the test  organism. If, how-
ever, the major mechanism is pathogenicity, a more appropriate  endpoint
would be overt symptomatology.

     (ii) The test data  should establish that the avian oral LD50, defined
as the  dose  required to kill  50  percent of the test organisms, or ID50,
defined as the  dose necessary to cause overt symptomatology in 50 percent
of the test organisms, are greater than the hazard dosage level. If the LD50
or ID50  is lower than the  maximum  hazard dose,  a definitive LD50  or
ID50 with confidence limits should be established.

     (10) Duration of test.  Control and treated groups should be observed
for at least 30 days after dosing initiation. If symptomatology  or  toxic
signs are manifest at the 30th day, observation should continue until recov-
ery, mortality,  or unequivocal moribundity is established.

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     (c) Reporting and evaluation of data. In addition to the information
specified in OPPTS  885.0001, the test report shall contain the following
information:

     (1) Species.

     (2) Age of the birds tested.

     (3) Mean body  weights  for each test and control group at test initi-
ation and weekly thereafter.

     (4) Diet analysis (especially antibiotics).

     (5) Pen dimensions.

     (6) Ambient temperature and humidity.

     (7) Photoperiod  and lighting.

     (8) Total feed consumption  for each test and control group at weekly
intervals.

     (9) Method of test material preparation, concentration of the MPCA
and total dose.

     (10) Amount of test material dosed per bird.

     (11) Amount of vehicle  dosed per bird, if a vehicle other than water
is used.

     (12) Number of birds per group.

     (13) LD50  or ID50 in appropriate units with 95 percent confidence
limits if obtained.

     (14) Methods used for calculation of LD50 or ID50.

     (15) Slope of the dose response line, if obtained.

     (16) Time and date of mortalities.

     (17) Any signs  of intoxication, abnormal behavior, and regurgitation
(if any occurs).

     (18)  Reports  of  any pathogenic  symptomatology  or  pathological
changes.

     (19) Results of  gross necropsies and histopathological findings con-
ducted on  enough birds to characterize any gross lesions  including  at-
tempts, using appropriate  techniques,  to  reisolate the  MPCA from exam-
ined tissues.

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     (d) Tier progression. (1) If any pathogenic symptoms or toxic signs
are observed at any dose level  in this  study, testing at Tier II, environ-
mental  expression  testing  (OPPTS   guidelines  885.5000,  885.5200,
885.5300, and 885.5400), is required as specified in 40 CFR 158.740. In
some cases, a  subchronic test may  serve to better the understanding of
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 Tier I study,
additional 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 birds despite the negative Tier I results.

     (e) References. The following references are provided for use in the
development of acceptable test protocols for conducting an avian oral path-
ogenicity/toxicity test with a microbial pest control agent.

     (1) Friend, M. and D.O. Trainer. Experimental duck virus hepatitis
in the mallard. Avian Disease 16:692-699 (1971).

     (2) Hartmann,  G.C. and S.S. Wasti. Avian safety of three  species of
entomogenous  fungi.  Comparative Physiological  Ecology   5:242-245
(1980).

     (3) Ignoffo, C.M. Effects of entomopathogens on vertebrates. Annals
New York Academy of Science 217:141-164 (1973).

     (4)   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).

     (5) Narayanan, K.  et al. Lack  of susceptibility of poultry birds to
nuclear polyhedrosis  virus  of groundnut red-hairy caterpillar, Amsacta
albistriga (W.). Indian  Journal of Experimental Biology.  16:1322-1324
(1978).

     (6) Podgwaite, J.D. and R.R. Galipeau.  Effects of nucleopolyhodrosis
virus an two avian predators of the gypsy moth.  USDA Forest Service
Research Notes, NE-251, 2 pp. (1978).

     (7) Summers,  M. et al., R. Engler,  L.A. Falcon,  and P.  Vail, eds.
Guidance   for   Safety  Testing  of Baculoviruses,   pp.   179-184   in:
Baculoviruses for Insect Post Control.  Safety Considerations.  American
Society for Microbiology, Washington, DC (1975).

     (8) Wolf, K. Evaluation of the exposure of fish and wildlife to nuclear
polyhedrosis and granulosis viruses, pp.  109-111 in: Baculoviruses for In-
sect Pest  Control. Safety Considerations.  American Society for Microbi-
ology, Washington, DC (1975).

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