United States Prevention, Pesticides EPA712-C-96-330
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.4100
Avian Inhalation Test,
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 885.4100 Avian inhalation test, 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-17. The Agency
recognizes that this test protocol has not yet been evaluated in the labora-
tory. An inhalation exposure may be acceptable in lieu of instillation and,
for some microorganisms, the Agency may accept a request for waiver
of this test with appropriate justification. It would be advisable to contact
the Agency before performing the respiratory testing.
(b) Test standards. Data on avian respiratory pathogenicity of micro-
bial pest control agents (MPCAs) must be derived from tests which satisfy
the purposes of the general test standards in OPPTS 885.0001, and all
the following test standards:
(1) Test substance. The actual form of the material to be used as
the test substance is described in OPPTS 885.4000. In addition, any sub-
stances used to enhance virulence or toxicity should be tested along with
the test substance.
(2) Species. Testing shall be performed on one avian species (pref-
erably bobwhite quail). Other species may be used but a justification must
be supplied based on increased susceptibility to the MPCA or ecological
considerations which preclude the use of recommended species.
(3) Age. Birds used in this test should be from 14 to 28 days old
at the beginning of the testing period. Within a given test, all birds shall
be as close to the same age as possible.
(4) Controls, (i) A negative, nondosed control group is required.
(ii) A concurrent control group is required and shall be treated with
the pure active ingredient that has been inactivated in such a way as to
preserve cellular integrity.
(iii) After dosing, two untreated contact control birds are required
and shall be placed in with the treatment group receiving the maximum
hazard dosage.
(5) Number of birds per dosage level. Each treatment and control
group shall contain at least 10 birds. When there is only one treatment
group at least 30 birds shall be tested at that treatment level.
(6) Route of exposure. The test material should be administered by
intranasal or intratracheal instillation. Depending on the physical properties
of the agent being tested, an alternate route, such as use of an aerosol,
-------
involving the respiratory tissues may be used. However, use of the alter-
nate route must be justified.
(7) Dosing regimen. Birds should receive doses daily for 5 days.
(8) Maximum hazard dosage level, (i) The maximum hazard dose
is defined by the following formula:
maximum daily dose (units) =
[MPCA] in TGAI x 0.2 mL/kg BW x weight of test bird (kg)
where
[MPCA] = concentration of MPCA
TGAI = technical grade of active ingredient
BW = body weight
(ii) For MPCAs that produce a toxin, fractions of this dose should
be calculated for lower doses. A reason shall be provided to support any
reduction in the highest dosage level.
(9) Treatment concentrations. 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)(10) of this guideline.
(10) Determination of an LD50 or ID50. (i) The study endpoint
must be defined to reflect the pathological activity of the specific agent
being tested, i.e. if an MPCA is expected to produce a toxin and has little
or no infectivity, the appropriate endpoint would be death of the test orga-
nism. If, however, the major mechanism is pathogenicity, a more appro-
priate endpoint would be overt symptomatology.
(ii) The test data must establish that the avian inhalation LC50, de-
fined as the dose required to kill 50 percent of the test organisms, or IC50,
defined as the dose necessary to produce overt symptomatology in 50 per-
cent of the test organisms, is greater than the maximum hazard dosage
level. If the LC50 or IC50 is less than the maximum hazard dose, a suffi-
cient number of treatment levels should be tested in order to obtain a defin-
itive LC50 or IC50, if possible.
(11) Duration of test. Control and treated birds should be observed
for at least 30 days after dosing. If symptomatology is manifest at the
thirtieth day, observation should continue until recovery, mortality, or un-
equivocal 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) Identification of vehicle or carrier used to disperse the agent and
the average amount of vehicle administered to each bird, if a vehicle other
than water is used.
(11) Number of birds per treatment level;
(12) Number of controls used;
(13) Time and date of mortalities or symptom onset;
(14) ID50 or LD50, in appropriate units, with 95 percent confidence
limits, if obtained.
(15) Results of gross necropsy and histopathological findings con-
ducted on all birds dying before termination of the test, on a representative
sample of those that survived, and on two contact control birds. The ne-
cropsy report should include any evidence of respiratory tract involvement
and involvement at distant sites including liver, kidney, spleen, cerebro-
spinal system, gastrointestinal system. Blood samples should also be ana-
lyzed for abnormalities. Results should also include any attempts, using
appropriate techniques, to reisolate the MPCA from examined tissues.
(16) A clinical assessment of any histopathological findings and le-
sions noted.
(17) Assessment of the clinical significance of MPCA tissue isola-
tions.
-------
(d) Tier progression. (1) If any pathogenic or toxic effects are ob-
served at the maximum hazard dosage level in this study, testing at Tier
II, environmental expression testing (OPPTS 885.5000, 885.5200,
885.5300, and 885.5400) is required as specified in 40 CFR 158.740(e).
In some cases, a subchronic test may serve to better understanding of ef-
fects observed at the Tier I level and alleviate the need for Tier II testing.
(2) If no pathogenic effects are observed in this study, no additional
testing at higher tiers ordinarily is required. The Agency may require addi-
tional 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 test protocols for conducting an avian inhalation patho-
genicity test with microbial pest control agents:
(1) Friend, M. and D.O. Trainer. Fob/chlorinated biphenyl: interaction
with duck hepatitis virus. Science 170:1314-1316 (1970).
(2) Friend, M. Experimental duck virus hepatitis in the mallard. Avian
Disease 16:692-699 (1971).
(3) Friend, M.. Duck hepatitis virus interaction with DDT and
Dieldrin in adult mallards. Bulletin of Environmental Contaminant Toxi-
cology 7:202-206 (1972).
(4) Friend, M. Experimental DDT-Duck hepatitis virus interaction
studies. Journal of Wildlife Management 38:887-895 (1974).
(5) Friend, M. Experimental Dieldrin-Duck hepatitis virus interaction
studies. Journal of Wildlife Management 38:896-904 (1974).
(6) Hartmann, G. C. and S. S. Wasti. Avian safety of three species
of entomogenous fungi. Comparative Physiological Ecology 5:242-245
(1980).
(7) 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).
------- |