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
-------
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."
-------
OPPTS 885.4050 Avian oral, 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-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.
-------
(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.
-------
(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.
-------
(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).
------- |