United States      Prevention, Pesticides     EPA712-C-96-318
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA   Microbial Pesticide
          Test Guidelines
          OPPTS 885.3200
          Acute Injection Toxicity/

     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 885.3200 Acute injection 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 the OPP guideline 152A-13.

     (b) Purpose.  Acute injection toxicity/pathogenicity data provide in-
formation on health  hazards likely to arise from a single exposure where
the skin is bypassed  as a barrier.  The purpose of the acute injection study
is to provide initial information on the toxicity, infectivity, and pathogenic-
ity of a MPCA using a single dose exposure and an adequate post-exposure
observation period.

     (c) Definitions.  The following definitions apply to this guideline.

     Acute injection  toxicity and acute injection pathogenicity are the ad-
verse effects occurring from the  intravenous or intraperitoneal administra-
tion of MPCA.  Acute injection  toxicity also  may be the adverse effects
occurring  from  the  intravenous or  intraperitoneal administration  of  a
microbially produced substance.

     Dose level  is  the amount of MPCA administered. It is expressed as
units of the microorganism administered per test animal.

     Units of MFC As 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

     (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 colony forming unit (CPU) on  appropriate
germination medium.

     (ii) Fungal  mycelium: 10'9 gm dry weight or, after standardized pre-
paratory procedures, a  mycelium-producing entity on  semisolid growth

     (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 CPU on an appropriate semisolid growth me-

     (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.

     (d) Principles of the test method. The test MPCA is  administered
by intravenous or intraperitoneal injection in a single high dose to experi-
mental animals. Subsequent observations of effects and deaths are made,
and, for  the  intravenous route only, the 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.
In general, smaller microorganisms (e.g. bacteria) should be  administered
by intravenous injection and larger microorganisms (e.g. fungi, protozoa)
should be administered by intraperitoneal injection.

     (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 physical  nature  of  some  technical grade preparations may
render injection testing impractical. In such cases, the  test substance may
consist only of the purest infective form of the microorganism. However,
justification/reasoning must be provided by the investigator  to support a
request for exempting specific technical grade preparations  from testing
by injection dosing.

     (3) 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  is
expected,  or if changes in form of the MPCA occur in  the host, then these
forms also may have to be tested.

     (f) Characteristics of the MPCA. The test MPCA  should  be thor-
oughly characterized as required in OPPTS 885.1000.

     (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 used. 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 nonpregnant.

     (ii) Age. Young adult animals should be used.  The weight 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

     (2) Control groups, (i) A concurrent untreated control group of two
animals/sex is required. A "shelf control group" is not required.

     (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. If included, inactivation should
be done by a means that allows for  reasonable maintenance of the struc-
tural integrity of the MPCA.

     (3) Dosing, (i)  Dose level. One dose level of at least 107  units of
the MPCA per test animal  should be used. If a dose level of at least 107
units per test animal is  not used, a justification/explanation must be pro-

     (ii) Vehicle. The recommended  vehicle is one that allows  for mainte-
nance of viability, or germination 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 by intravenous or intraperitoneal injection at one time depends on
the size of the test animal. 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 via  a
needle and syringe in a single dose.

     (ii) If a single dose is not possible, the dose may be given in smaller
fractions over a period not exceeding  24 h.

     (5) Observation period. The observation period should  be at least
for 21 days after dosing. However, the duration of observation  should not
be fixed rigidly. It should be determined by the type of MPCA adminis-
tered and, when determined, its rate of clearance from the test animals.
Duration of the observation period also would depend on the time at which
signs of toxicity and pathogenicity 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,
changes 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) Individual weights of 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 one 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. All gross pathological changes should be recorded.

     (8) Clearance of MPCA: Intravenous injection. Blood  from test
animals should be collected during the study and examined for the pres-
ence of the MPCA to estimate clearance of the MPCA after dosing. The
first  analysis  should be  done on three animals per  sex as soon as reason-
ably possible  after dosing. 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. Determina-
tions of clearance are not required after intraperitoneal injection.

     (9) MPCA enumeration in tissues, organs, and body fluids: Intra-
venous injection.  Infectivity and clearance  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 selected for MPCA enumeration should, if possible, allow
for detection of microbial replication. MPCA enumeration in tissues, or-
gans,  and body fluids is not required after intraperitoneal injection.

    (10) Interim sacrifice:  Intravenous injection  only. 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 then at 1 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  adequately establish a pattern
of clearance.

    (11) Tissues, organs,  and body fluids: Intravenous injection  only.
(i) For infectivity and persistence determinations, the MPCA should be
enumerated from the kidneys, brain, liver, lung, spleen, blood, representa-
tive lymph nodes, caecum  contents, and, where appropriate,  from lesions
and the inoculation 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 patho-

    (iv) Description of toxic and pathogenic effects.

    (v) Definition for one  unit of the MPCA used, and the units per test
animal in the dose.

    (vi) Body weights and  time taken.

    (vii) Necropsy findings.

    (viii) Pathology findings, when performed.

    (ix) Infectivity findings (intravenous injection only).

    (x) Estimate of  rate of MPCA clearance (intravenous injection  only).

    (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) Pathogenicity.
     (i)  Tier progression.  (1) If persistent or significant signs of patho-
genicity of the MPCA for the  test animals is  observed in Tier I, acute
injection toxicity/pathogenicity tests may be required in nonrodent  animal
species. Consultation with the Agency is recommended to determine the
requirement for any further appropriate testing.
     (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 pathogenicity:
     (i)  Toxic components of the dosing material are to be identified, and
where possible, isolated.
     (ii)  An  acute toxicity  study, (OPPTS  885.3550) is to be conducted
with the toxic components.