United States Prevention, Pesticides EPA712-C-96-349
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.7200
Domestic Animal Safety
'Public Draft"
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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.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
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), 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 870.7200 Domestic 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) Domestic animal safety studies are intended to dem-
onstrate that pesticide formulations for the treatment of external pests on
domestic animals are safe under conditions of recommended use and that
an adequate margin of safety exists if the products are misused (overused).
Data from domestic 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 requirements for these studies.
Although not a toxicity study of the type required for pesticide registration,
a domestic 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 domestic 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 guidelines address data requirements for the safety assessment
of products applied directly to animals. Products used to treat external
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 depend-
ent on label claims. Labeled uses also impact on the duration of treatment
and on the age and species of test animal used in a domestic animal safety
study.
(5) The studies conducted to satisfy these guidelines should not be
mistaken for toxicity studies, as their intent is not to establish a no-ob-
served-effect-level (NOEL), but to provide assurance that a adequate mar-
gin of safety exists.
(6) Studies conducted to satisfy domestic animal safety guidelines
should be conducted in compliance with 40 CFR part 792 and 40 CFR
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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 domestic animal safety stud-
ies, the test substance is always the final formulation of a pesticide prod-
uct.
Adverse effect is an undesirable effect reflected in the animal by alter-
ations in structure, function, or behavior.
Domestic animal will be limited to dogs and cats for the purpose
of this guideline.
Domestic 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 domestic animal safety study
guidelines do 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 domestic animal
safety study should reflect the product label, i.e. the method of administra-
tion, species and age group, frequency of application, etc., used in the
study should be identical to that of the end-use product. The test formula-
tion should be applied to several groups of experimental animals at the
label recommended dose and multiples of this dose (3x and 5x the rec-
ommended dose).
(2) If the product label states that a treatment can be repeated, the
domestic animal safety study should also include a repeat treatment.
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(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 (lx). For exaggerated doses (3x and 5x the rec-
ommended dose), products specifically prepared for this type of study that
contain higher concentrations (3x and 5x) of the active ingredient are pre-
ferred. See additional discussion under paragraph (g)(3)(iii) of this guide-
line.
(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 young 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 domestic animal
safety study.
(iii) Sex. (A) Equal numbers of animals in 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
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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. (This is optional.)
(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-
able.
(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 domestic animal safety study. When repeat
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treatments are required, products will be handled on a case-by-case basis
with the retreatment interval being driven by label claims and instructions
for use. 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-
uct.
(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-
tions.
(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.
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(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
membranes.
(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
time.
(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 adults and juveniles should be separated.
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(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, and identified target organ, 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 160, subpart J, the test report summary
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
termination.
(B) Time of observation of each abnormal sign and its subsequent
course.
(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.
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(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.
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