United States Prevention, Pesticides EPA712-C-96-315
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.3050
Acute Oral Toxicity/
Pathogenicity
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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 885.3050 Acute oral toxicity/pathogenicity.
(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 152A-10.
(b) Purpose. In the assessment and evaluation of the toxic or patho-
genic characteristics of an MPCA, determination of acute oral toxicity/
pathology usually is an initial step. It provides information on health haz-
ards likely to arise from a single exposure by the oral route. The purpose
of the acute oral study is to provide initial information on the toxicity,
infectivity, and pathology of a MPCA using a single high dose exposure
and an adequate post-exposure observation period.
(c) Definitions. The following definitions apply to this test guideline.
Acute oral toxicity and acute oral pathogenicity are the adverse ef-
fects occurring from the oral administration of a single dose of a MPCA.
Acute oral toxicity also may be the adverse effects occurring from the
oral administration of a microbially produced substance, or from other in-
gredients in any test substance.
Dose level is the amount of MPCA administered. It is expressed as
units of the microorganism administered per test animal.
Units of MPCAs One unit of representative MPCA groups usually
would be defined as follows: (Due to the wide diversity of forms among
microorganisms, other definitions of a unit of a MPCA may be equally
appropriate.)
(i) Bacterial or fungal spore, bacterial or protozoan cyst: An intact
individual spore or cyst as determined microscopically, and usually the
entity that produces a single CPU on appropriate germination medium.
(ii) Fungal mycelium: 10-9 gram dry weight or, after standardized pre-
paratory procedures, a mycelium-producing entity on semisolid growth
medium.
(iii) Protozoa: An intact vegetative organism, spore, or cyst of the
members in the various classes of this phylum.
(iv) Vegetative bacterium: A single, viable organism, and usually the
entity that produces a single colony forming unit (CPU) on an appropriate
semisolid growth medium.
(v) Virus: An intact, complete virion or a polyhedral body as deter-
mined by microscopy, and, usually the entity that produces an infective
unit (IU) on appropriate host cells or tissues.
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(d) Principles of the test method. The test MPCA is administered
orally by gavage in a single high dose to experimental animals. Subsequent
observations of effects and deaths are made and rate of clearance of the
MPCA is estimated. Animals that die during the test are necropsied, and
at the conclusion of the test the surviving animals are sacrificed and
necropsied. Infectivity of the MPCA is evaluated periodically during the
test, and at the conclusion of the test.
(e) Substance to be tested. (1) The technical grade of each active
ingredient shall be tested to support the registration of each manufacturing-
use product and each end-use product.
(2) The form (e.g. vegetative cell, spore, cyst, virion) of the MPCA
used in testing should be equivalent to the form that is intended for reg-
istration or application. To the extent possible, the test MPCA also should
be equivalent to the MPCA intended for registration or application with
respect to stage of growth, possession of organelles and appendages and
expression of phenotypic traits (including products from intentionally in-
troduced genes). If significant exposure to other forms of the MPCA are
expected, or if changes in form of the MPCA occur in the host, these
forms also may have to be tested.
(f) Characteristics of the test MPCA. The test MPCA should be
thoroughly characterized as required in Group A of this series of guide-
lines—Product Analysis Test Guidelines.
(g) Test procedures—(1) Animal selection—(i) Species and strain.
Although several mammalian test species may be used, the mouse or the
rat are the preferred rodent species. Commonly used laboratory strains
should be employed. If another species is used, justification/reasoning for
the alternative selection should be provided. All test animals should be
free of parasites or pathogens. Females should be nulliparous and nonpreg-
nant.
(ii) Age. Young adult animals should be used. The weight variation
of animals used in a test should not exceed ±20 percent of the mean
weight for each sex.
(iii) Sex. Equal numbers of animals of each sex are required.
(iv) Numbers. At least six animals (three animals of each sex) should
be used. A sufficient number of additional animals should be used to allow
for interim sacrifice for infectivity determinations, and also, for control
groups.
(2) Control groups, (i) A concurrent untreated control group of four
animals per sex is required. Half of the animals in the control group (i.e.,
two animals per sex) should be housed separately from the test group of
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animals dosed with MPCA, and the remainder of the control animals (the
"shelf control" group) should be housed with the dosed animals.
(ii) A separate vehicle control group is not required except when the
toxicity of the vehicle is unknown.
(iii) Control groups dosed with inactivated MPCA (i.e., rendered in-
capable of reproduction or germination or excystment) may prove useful
to evaluate toxic properties of the MPCA. Inactivation should be done
by a means that allows for reasonable maintenance of the structural integ-
rity of the MPCA.
(3) Dosing—(i) Dose level. One dose level of at least 108 units of
the MPCA per test animal should be used. If a dose level of at least 108
units per test animal is not used, a justification/explanation must be pro-
vided. The test material should not be diluted to reach the limit dose at
108 units per test animal.
(ii) Vehicle. The recommended vehicle for the technical grade of each
active ingredient is one that allows for maintenance of viability, or germi-
nation capability, or excystment capability, or, for intracellular parasites,
infection capability in a suitable host.
(iii) Volume. The maximum volume of liquid that can be adminis-
tered at one time depends on the size of the test animal. In rodents, the
volume should not exceed 2 mL/100 g body weight. Variability in test
volume should be minimized.
(iv) Dose quantification. Techniques used to quantify the units of
MPCA in any dose will depend on the group of microorganisms to which
the MPCA belongs. Where possible, determinations of viable, or poten-
tially viable, or infective units in each dose should be made. A measure-
ment of metabolism associated with a defined biomass may be the pre-
ferred technique for quantification of mycelial forms of MPCAs. Quan-
tification should be done concurrently with testing.
(4) Exposure, (i) The test substance should be administered in a sin-
gle dose by gavage, using a stomach tube or suitable intubation cannula.
(ii) Animals should be fasted overnight prior to test substance admin-
istration. After the substance has been administered, food may be withheld
for a further 3-4 h.
(iii) If a single dose is not possible, the dose may be given in smaller
portions over a period not exceeding 24 h. Where a dose is administered
in fractions over a period, it may be necessary to provide the animals
with food and water, depending on the length of the period.
(5) Observation period. The observation period should be at least
for 21 days after dosing. However, the duration of observation should not
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be fixed rigidly. It should be determined by the type of MPCA adminis-
tered and its rate of clearance from the test animals. Duration of the obser-
vation period also would depend on the time at which signs of toxicity
and pathology appear and disappear, and the time to death of the animals.
(6) Observation of animals, (i) A careful clinical examination of all
animals should be made at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g. necropsy of,
and MPCA enumeration from those animals found dead, and isolation of
weak or moribund animals.
(iii) Cageside observations should include, but not be limited to,
change in:
(A) The skin and fur.
(B) Eyes and mucous membranes.
(C) Respiratory system.
(D) Circulatory system.
(E) Autonomic and central nervous system.
(F) Somatomotor activity.
(G) Behavior pattern.
(H) Particular attention should be directed to observation of tremors,
convulsions, diarrhea, lethargy, salivation, sleep and coma.
(iv) Weights of individual animals should be determined shortly be-
fore the test material is administered, weekly thereafter, and at death or
at interim or final sacrifice. Changes in weight should be calculated and
recorded when survival exceeds 1 day.
(v) The time of death should be recorded as precisely as possible.
(7) Gross pathology. A gross necropsy of all animals should be per-
formed at the time of death or at interim or final sacrifice. All gross patho-
logical changes should be recorded.
(8) Clearance of MPCA. Feces from test animals should be collected
soon after dosing and frequently during the study and examined for the
presence of the MPCA to estimate clearance of the MPCA after oral ad-
ministration. Methods (e.g. immunological assays, DNA probes) other than
those used for quantification of MPCAs in each dose may prove useful.
Recovery values and detection and sensitivity limits should be determined
and reported for each technique used.
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(9) MPCA enumeration in tissues, organs, and body fluids. Infec-
tivity or persistence should be assessed by using sensitive techniques to
determine the presence of the MPCA in tissues, organs, and body fluids.
Methods other than those used for quantification of MPCAs in each dose
may prove useful. Recovery values and detection and sensitivity limits
should be determined and reported for each technique used. Methods se-
lected for MPCA enumeration should, if possible, allow for detection of
microbial replication.
(10) Interim sacrifice. For evaluating infectivity and clearance, the
MPCA should be enumerated from tissues, organs, and body fluids of three
treated animals per sex, sacrificed at 3 days after, and at one week intervals
after dosing. The number of interim sacrifice periods required will depend
on the nature of the test microorganism, and should be sufficient to estab-
lish a pattern of clearance adequately. The MPCA also should be enumer-
ated from the tissues, organs, and body fluids of the "shelf control" group
at final sacrifice.
(11) Tissues, organs, and body fluids, (i) For infectivity and persist-
ence determinations, the MPCA should be enumerated from the kidney,
brain, liver, lung, spleen, blood, representative lymph nodes, and, where
appropriate, from lesions and from the injection site.
(ii) Other tissues, organs, and body fluids may have to be examined
as indicated by the nature of any toxic and pathogenic effects observed.
(h) Data and reporting—(1) Treatment of results. In addition to
the information recommended by OPPTS 885.0001, the test report should
include the following information:
(i) Number of animals at the start of the test.
(ii) Time of death of individual animals.
(iii) Number of animals displaying other signs of toxicity and pathol-
ogy.
(iv) Description of toxic and pathogenic effects.
(v) Definition for one unit of the MPCA used, and the units/test ani-
mal in the dosing material.
(vi) Body weights and time taken.
(vii) Necropsy findings.
(viii) Pathology findings, when performed.
(ix) Infectivity/persistence findings.
(x) Estimate of rate of MPCA clearance.
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(xi) Description of all enumeration methods used for MPCA detection
and quantification.
(xii) Verification that each enumeration method is sufficiently sen-
sitive to serve as a useful quantitative assay, for the MPCA in tissues,
organs, and body fluids.
(2) Evaluation of results. An evaluation should include the relation-
ship if any, between exposure to the test substance and the incidence and
severity of all abnormalities, including:
(i) Behavioral abnormalities.
(ii) Clinical abnormalities.
(iii) Gross lesions.
(iv) Body weight changes.
(v) Mortality.
(vi) Toxicity.
(vii) Infectivity.
(viii) Pathology.
(i) Tier progression. (1) If persistent or significant signs of pathology
of the MPCA for the test animals is observed in Tier I, acute oral toxicity/
pathology tests may be required in nonrodent animal species. Consultation
with the Agency to discuss further testing requirements is recommended.
(2) If toxin production by the MPCA is suspected, or if toxin produc-
tion is indicated by significant or persistent signs of toxicity in the test
animals, in the absence of signs of infectivity or pathology;
(i) The toxic component(s) of the dosing material is (are) to be identi-
fied, and to a practical extent, isolated.
(ii) An acute toxicity study (OPPTS 885.3550) is to be conducted
with the toxic component(s).
(3) If significant infectivity or unusual persistence of the MPCA is
observed in the absence of signs of toxicity or pathogenicity, a subchronic
(90-day) study (OPPTS 885.3600) is required.
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