United States       Prevention, Pesticides     EPA712-C-98-349
          Environmental Protection    and Toxic Substances     August 1998
          Agency         (7101)
&EPA    Health Effects Test
          OPPTS 870.7200
          Companion Animal

     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 disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.7200  Companion animal safety.
     (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 81-6 Domestic Animal  Safety Test-
ing (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals)  EPA report 540/09-82-025, 1982.

     (b) Purpose. (1) Companion animal safety studies are  intended  to
demonstrate that pesticide formulations for the treatment of external pests
on domestic animals  have an adequate margin of  safety  if the products
are misused (overused). Data from companion animal safety  studies also
serve as a basis for product labeling. This guideline is intended to promote
uniform review of data and to  assure consistency  and fairness in the re-
quirements for these  studies. Although not a toxicity study  of the type
required for pesticide registration, a companion animal safety study is most
comparable to an acute dermal toxicity study.

     (2) This guideline also serves the purpose of providing harmonization
between the Environmental Protection Agency and  the Center for Veteri-
nary Medicine in the Food and  Drug Administration (FDA), which is also
responsible for conducting target animal safety studies.

     (3) This guideline is limited  to products for  use on dogs and cats
due to  the high volume use of products in these two species. The guidance
is  based on professional experience, documentation in  the scientific  lit-
erature, and policy and procedures of other agencies involved in regulatory
veterinary medicine,  (see Target Animal Safety Guidelines for New Ani-
mal Drugs, under paragraph (i)(3) of this guideline.)

     (4) The guideline addresses data requirements for the safety assess-
ment of products applied directly to animals.  Products used to treat exter-
nal pests  on domestic  animals include, but are not limited to  collars,
sprays, dips, shampoos, and spot treatments. Due to differences in methods
of application, specific testing procedures for individual products are de-
pendent on label claims. Labeled uses also impact on the duration of treat-
ment and on the age and species of test animal used in a companion animal
safety study.

     (5) The studies conducted to satisfy this  guideline should not  be mis-
taken for toxicity studies, as their intent is not to establish a no-observed-
effect-level (NOEL), but to provide assurance that  an adequate margin  of
safety exists.

     (6) Studies  conducted to satisfy companion animal safety guidelines
should be conducted  in compliance with 40  CFR  part 792 and 40 CFR

160 (Good Laboratory Practice Standards) and a statement of compliance
should be contained within the final report.

    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
part 792 apply to this test guideline. The following definitions also apply
to this test guideline.

    Acute dermal toxicity is the adverse effect occurring during or follow-
ing a dermal  exposure to a single dose of a test substance or  to  repeat
applications to achieve dose.  For purposes of companion animal  safety
studies, the  test substance is always the final formulation of a pesticide

    Adverse effect is an undesirable effect reflected in the animal by alter-
ations in structure, function, or behavior.

    Companion animal will be limited to dogs and cats for the purpose
of this guideline.

    Companion animal safety study is a study conducted for the purpose
of establishing an adequate margin of safety and not a NOEL of toxicity.

    Dosage is a term comprising the dose, its frequency, and the duration
of dosing.

    Dose is the  amount of test substance administered and is expressed
in weight of test  substance per unit weight  of the test animal (e.g. milli-
grams per kilogram).

    Margin of safety  is the difference between  the effective dose (rec-
ommended dose)  and the  toxic dose. The companion animal safety study
guideline does not require the determination of a toxic dose but  rather
the establishment  of an adequate margin of safety.

    Max/tox is a product containing multiple toxicants at the maximum
levels which would be anticipated in formulated topical products.

    Vehicle is the end-use product formulation, i.e. the inert ingredients
without the active ingredients.

    (d)  Principle of the test method. (1) The  design of a companion
animal safety  study should  reflect the  product label, i.e. the method of
administration, species and age group, frequency of application, etc., used
in the study should  be identical to that of the end-use product.  The test
formulation  should be  applied to several groups of experimental animals
at the label recommended dose and multiples of this dose (3X and 5X
the recommended dose).

    (2)  If the product label states that a treatment can be repeated, the
companion animal safety study should also include a repeat treatment.

     (3) Observations and measurements of possible treatment-related ef-
fects should be reported for 14 days posttreatment. Animals  that die or
are sacrificed  in a moribund state should be  subjected to a necropsy in
an attempt to  arrive  at the cause of death. Routine sacrifice or necropsy
is not required for surviving animals.

     (e) Substance to be tested. (1) The end-use product should be tested
at  the recommended dose (IX). For exaggerated doses (3X and 5X the
recommended  dose), products specifically prepared for this type of study
that  contain higher concentrations (3X and 5X) of the active ingredient
are preferred. See additional discussion under  paragraph (g)(3)(iii) of this

     (2) Because  of the practice of  combining several pesticides in one
product, a procedure has been proposed whereby maximum concentrations
of multiple active ingredients have been used  to determine the margin of
safety of end-use  products. This practice has been referred to as the max/
tox procedure.

     (f)  Limit test.  If a test  at one dose level of at least 5X the rec-
ommended dose,  using the procedures described for the study, produces
no evidence of treatment-related toxicity, a full study using a minimum
of three dose levels may not be necessary.

     (g) Test procedures—(1) Animal  selection—(i) Species. The species
recommended  for treatment on the product label  should be included in
the study. Studies should be performed  on healthy dogs and cats represent-
ative of the classes of dogs  and cats (size, weight  range, sex,  or  age) for
which the product is intended.

     (ii) Age.  The age of animals in the study is  dependant  upon label
claims. If only adults (6 months or older) are the targeted population of
animals to receive treatment, adults only will suffice. However, if a prod-
uct is registered for use on pediatric  animals (i.e. puppies and  kittens),
the label should state a minimum age  for this group, for example,  "Do
not use on puppies (kittens)  less than eight weeks of age". Consequently,
the product should be tested in 8 week-old animals  in the  companion ani-
mal safety study.

     (iii) Sex. (A) Equal numbers of animals of each sex are recommended
for each dosage level.

     (B) Females should be nonpregnant.

     (iv) Numbers. At least six animals per sex should  be used at  each
dosage level.

     (v) Pretreatment. Animals should be vaccinated, dewormed, and ac-
climated for 2 weeks prior to the initiation  of the  study. They should be
examined by a veterinarian and their suitability should be ascertained prior

to inclusion in the study. Animals should be free of infectious diseases
which could complicate the interpretation of the study results.

     (2)  Control  group. A  concurrent vehicle  control  group  is  rec-
ommended. Negative (untreated) controls may occasionally be  employed
to determine whether  adverse effects are due to the inert ingredients  in
a formulation.

     (3) Dosing—(i) Dose levels. The dose levels of the end-use product
to be tested should include control (OX), IX, 3X, and 5X the recommended
dose. The targeted adequate margin  of safety is  5X. Consideration will
be given to products with less than a 5X margin of safety, depending on
the severity of clinical signs of toxicity (e.g. transient, non-life-threatening
signs). The  route  of administration  should be the proposed  label route.

     (ii) Vehicle. The vehicle control should be administered at a 5X level.
The vehicle should contain the  inert ingredients at the maximum levels
that would appear in the 5X formulation.

     (iii) Methods  of achieving exaggerated  doses. It is preferred that
formulations be revised to include exaggerated amounts of the active ingre-
dient. However, if this cannot be achieved because of volume constraints
or the physical properties of the ingredients (active or inert), multiple treat-
ments at frequent intervals during the same dosing period will be accept-

     (A) Spray,  dip or shampoo formulations may be applied at the rec-
ommended dose at hourly intervals  to  achieve  an exaggerated  dose, i.e.
three times for a 3X dose and five times for a 5X dose. Other methods
to achieve exaggerated doses will be considered on a case-by-case basis.

     (B) Multiple collars may be worn simultaneously by the experimental
animals to achieve an exaggerated dose.

     (4) Observation  period. The observation period should be at least
14  days following the last treatment. The time at which clinical signs  of
toxicity appear and disappear should be recorded. The duration of the ob-
servation period should not be rigid, but should be determined by the toxic
reactions, the rate of onset and the length of the recovery period.

     (5) Administration, (i) The route of administration for these products
should be by the topical or dermal route. The product should be applied
in accordance with the label directions. No clipping of the hair or prepara-
tion of the skin is required  unless  such directions appear on  the label.

     (ii) If a single dose  is not possible, multiple  treatments at frequent
intervals during the same dosing period will be acceptable.

     (iii) If the product label recommends repeat treatments, multiple treat-
ments should be included in a companion animal safety study.  When re-

peat treatments are required, products will be handled on a case-by-case
basis with the retreatment interval being driven by label claims and instruc-
tions for use. For fleas collars, a  one month duration  of study may be
sufficient for well-characterized products. Several months may be required
for new chemical entities or collars containing multiple active ingredients.
Exceptions would include:

     (A) Single ingredient diluted products intended for use on dogs only,
where chronic studies in the intended species exist with the technical prod-

     (B) Products with retreatment intervals of 30 days or more.

     (C) Products with retreatment intervals  of 14 to 30 days which have
no observed toxicity following exposure to a 5X dose level.

It is advisable that when complex  issues arise, protocols be submitted in
advance for review and comment.

     (6) Observations of animals,  (i) Careful clinical observations should
be  conducted at hourly intervals  on the day of treatment for  at  least 4
h after  the last  treatment and twice daily thereafter  for the duration of
the observation period.

     (ii) If adverse reactions are observed, the observation period on the
day of treatment should be extended to a time at  which no further adverse
reactions are  observed.

     (iii) Observations  should include, but not be limited to, changes in
skin and fur, eyes  and mucous membranes, respiratory system, circulatory
system, autonomic and central nervous system, somatomotor activity,  and
behavior pattern. Particular attention should be  directed to observations
of central nervous  system signs (seizures, tremors, salivation),  vomiting
and diarrhea.

     (iv) The following provides a more  complete list of possible observa-

     (A)  Ocular:   Corneal  opacities,  nystagmus,   pupillary   changes,
blepharo spasm,  blindness,  iritis,  chemosis,  photophobia,  congestion,
blanching, discharge, and conjunctivitis.

     (B) Equilibrium: Unsteadiness (walking or standing), incoordination,
ataxia or paresis, and abnormal reflexes.

     (C) Muscular disturbances:  Localized or  generalized tremors,  lip
drooping and/or salivation, paralysis, atony, and atrophy.

     (D) Behavior (mental attitude): Anxious, apprehensive, circling, co-
matose, depressed, sedated, restless, panting, convulsions, and aggression.

     (E) Integument: Alopecia, haircoat condition, status of hydration, pru-
ritus, and erythema.

     (F) Gastrointestinal: Consistency of stools, propulsive diarrhea, hyper-
active gut, abdominal muscle tenseness, and vomiting.

     (G)  Cardiovascular: Heart rate, heart rhythm, and color of mucous

     (H)  Appetite/general health:  Body weight,  feed consumption, and
water consumption.

     (I) Respiratory:  Respirations/minute, dyspnea,  respiratory  sounds,
color of mucous membranes, nasal discharge, apnea.

     (v) Individual body weights should be measured twice during the ac-
climation period,  with the second  measurement being taken  immediately
prior to the initiation of treatment,  and on day-7 and day-14 of the obser-
vation period.

     (vi) Individual food consumption should be measured on a daily basis.

     (vii) The  time of death should be reported for animals dying or sac-
rificed moribund. Gross necropsies should be conducted to determine the
cause of death; abnormal organs/tissues should be  examined histologically.

     (viii) Clinical pathology should be assessed  prior to treatment, 24 h
posttreatment  and, if altered, on day-7.  Clinical  pathology  examinations
are required to determine the possibility of a treatment-related effect on
hematology and clinical chemistry parameters, regardless of whether  or
not  clinical signs of toxicity are  evident.  The  following  examinations
should be made:

     (A)  Hematology; Hemoglobin, mean corpuscular volume,  hemato-
crit,mean corpuscular hemoglobin,  red blood cell  count, mean corpuscular
hemoglobin concentration, white blood cell count, white blood  cell dif-
ferential  count, prothrombin time, and activated partial thromboplastic

     (B) Clinical  chemistry.  Glucose,  creatinine,  sodium, chloride, potas-
sium, phosphorus, total protein, albumin, globulin, calcium, blood urea ni-
trogen, alkaline phosphatase,  aspartate aminotransferase  (AST), alanine
aminotransferase (ALT), and total and direct bilirubin.

     (C) If the compound is a known cholinesterase inhibitor, plasma, and
red cell cholinesterase should be examined pretreatment, and at 6 to 8,
24, and 48 h, posttreatment.

     (h) Data  and reporting—(1) Treatment of results,  (i) Data should
be summarized in tabular form, showing, for each test group:

     (A) The number, age and sex of the animals at the start of the study.
Results from adult and pediatric animals should be separated.
     (B) The number of animals  displaying clinical  signs of toxicity, a
description of the observations and the time of onset.
     (C) Individual and group mean values  for body weight,  food  con-
sumption,  and clinical pathology measurements.
     (D) Time and  cause  of death, if known, for animals  which died or
were sacrificed in a moribund condition.
     (ii) All observed results should be evaluated by an appropriate statis-
tical method that was selected during the design of the  study.
     (2) Evaluation of study results. The evaluation should include the
relationship between the dose of the test substance and the presence or
absence, the incidence and severity, of abnormalities, including behavioral
abnormalities, clinical abnormalities,  body weight changes, effects on mor-
tality and  cause of death, identified target organ, and  any other general
or specific acute toxic effect.
     (3) Test report. In addition to information required under 40 CFR
part 792,  subpart J, and 40  CFR part 160, subpart J,  the test report sum-
mary should include the following information.
     (i) Toxic response and other effects data by sex and dose.
     (ii) Species and breed used.
     (iii) Individual animal data for the following:
     (A) Time of death during the study  or whether  animals survived to
     (B) Time of observation of each abnormal sign and  its  subsequent
     (C) Food consumption.
     (D) Body weight data.
     (E) Hematology tests  employed and the results.
     (F) Clinical chemistry tests employed and the results.
     (G) Necropsy  findings  on animals that died or were  sacrificed in a
moribund  condition.
     (H) Detailed description of histological findings on animals that died
or were sacrificed in a moribund condition.
     (I) Statistical treatment of results.

     (J) Other observations and/or findings.

     (i) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1) Hoskins, J.D.  Veterinary Pediatrics.  Dogs and Cats From Birth
to Six Months. Saunders, Philadelphia (1990).

     (2) Jones, B.D. Liver  and Pancreatic Disease. Presented to  B.C.
Academy of Veterinary Medicine, Fairfax, VA, June 9, 1994.

     (3) Target Animal Safety Guidelines for New Animal Drugs. Prepared
by the  Office of New Animal Drug Evaluation, Center for Veterinary Med-
icine, Food and Drug Administration, June 1989.