United States       Prevention, Pesticides     EPA712-C-96-316
          Environmental Protection    and Toxic Substances     February 1996
          Agency        (7101)
&EPA   Microbial Pesticide
          Test Guidelines
          OPPTS 885.3100
          Acute Dermal 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 885.3100  Acute dermal toxicity/pathology.
     (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-11.

     (b) Purpose. Acute dermal  toxicity data provide information on
health hazards likely to arise from a single dermal application of soluble
or particulate chemicals associated with a preparation of the MPCA, and/
or associated with other ingredients in formulations of the MPCA, and/
or associated with products from genetic  material intentionally introduced
into the MPCA.

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

    Acute dermal toxicity is the adverse effect occurring during or follow-
ing a 24-hour dermal exposure to a single dose of a test substance.

     (d) Principles  of the test method.  The MPCA in each formulation
to be tested is applied in a  single high dose to the skin of experimental
animals. Subsequently, observations of effects and deaths are made. Ani-
mals that  die during the  test  are necropsied, and at the conclusion of the
test,  the surviving animals are  sacrificed  and  necropsied as indicated by
the nature of the toxic effects  observed.

     (e) Substance to be tested. (1) The manufacturing-use product shall
be tested  to support the registration of each manufacturing-use product.

     (2) The end-use product shall be tested to support the  registration
of each end-use product.

     (f) Test procedures—(1) Animal selection—(i) Species and strain.
Although  several mammalian test species may be used, the albino  rabbit
is the preferred species. Commonly used laboratory strains  should be em-
ployed. If another species is used, the investigator should provide justifica-
tion/reasoning for the alternative selection. 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 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 with healthy  intact
skin  are recommended.

     (iv) Numbers.  At least 10 animals (5 animals of each sex) should
be used.

     (2) Control groups. Neither a concurrent untreated nor vehicle con-
trol group are required except when the toxicity of the vehicle is unknown.

     (3) Dosing—(i) Dose  level. The test substance should be applied at
2 g/kg BW. If a dose level  of less than 2 g/kg BW is used, a justification/
explanation must be provided.

     (ii) Vehicle. Where necessary, the  formulation  to be  tested is sus-
pended in  a suitable vehicle.  An aqueous solution  should  be used. The
recommended vehicle for the end-use product usually is the  same material
in which the MPCA will be mixed, suspended, or diluted for application.

     (iii) Volume. The  moisture  content of the test material should not
be excessive, but should be sufficient to prevent significant drying of the
test material during the  exposure period, and to ensure good contact with
the skin.

     (4) Exposure duration. The exposure duration should be for approxi-
mately 24 h.

     (5) Observation period. The  observation period should be at least
for 14 days, but should not be fixed rigidly. It should be determined  by
the toxic reactions, rate of onset, and length  of recovery period and may
be extended when considered necessary.  The time at which toxicity signs
appear and disappear, and their duration are important.

     (6) Preparation of animal skin, (i) Approximately 24 h before the
test, fur should be removed from the dorsal and ventral area of the trunk
of each test animal by clipping or shaving.

     (ii) Not less  than  10 percent of the body  surface  area should  be
cleared for application  of  the test  substance. The  weight of the animal
should be  taken into account when deciding on the area  to be  cleared
and on the  dimensions of the covering.

     (7) Application of the test substance (i) The test substance should
be applied uniformly over  an area  which is approximately  10 percent of
the total body surface area.

     (ii) The test substance should  be held in contact with the  skin with
porous gauze and a nonirritating tape throughout a 24-h exposure period.
The  test site further should be covered in a suitable manner to retain the
gauze dressing and test substance and ensure that the animals cannot ingest
the test substance.  Restrainers may be  used to  prevent the ingestion of
the test substance, but complete immobilization is not recommended.

     (iii) At the end of the  exposure period, residual test substance should
be removed, where practical, using water.

     (8) Observation of animals, (i) A careful clinical examination should
be made at least once each day.

     (ii)  Cageside observations should include,  but not be  limited  to,
changes in:

     (A) The skin (including signs of irritation) 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.

     (iii) Individual weights of animals should be determined shortly be-
fore the test  material is  administered, weekly thereafter, and  at death or
at final sacrifice. Changes in weight should be calculated  and recorded
when survival exceeds 1 day.

     (iv) The time of death should be recorded as precisely as possible.

     (v) At the  end of the 24-h exposure period, and daily thereafter, any
signs of skin irritation should be recorded and scored.

     (9) Gross  pathology. Consideration should  be given to  performing
a gross necropsy of all  animals if indicated by the appearance of toxic
effects. If done, all gross pathological changes should be recorded.

     (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.

     (iv) Description of toxic effects.

     (v) Definition for one unit of the MPCA used, and the units  in the
dosing material.

     (vi) Dry weight and net weight determinations of the test  material
applied per kilogram body weight of the test animal.
     (vii) Body weights and time taken.
     (viii) Necropsy findings, when performed;and,
     (ix) Pathology findings, when performed.
     (2) Evaluation of results. An evaluation should include the  relation-
ship if any,  between the animals exposed to  the test substance  and the
incidence and severity of all abnormalities, including:
     (i) Behavioral abnormalities.
     (ii) Clinical abnormalities.
     (iii) Skin lesions and skin irritation.
     (iv) Body weight changes.
     (v) Mortality.
     (vi) Toxicity.
     (i) Tier progression (1) If evidence of significant and/or persistent
toxicity are observed, toxic components of the dosing material are to be
identified, and to a practical extent, isolated.
     (i) An acute toxicity study (OPPTS 885.3550) is to be conducted with
the toxic components.
     (ii) [Reserved]
     (2) If no toxic effects are observed, no  further testing is  required.