United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–349

June 1996

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized OPPTS test guideline is OPP 81–6 Domestic Animal Safety Testing (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 demonstrate 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 Veterinary 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 literature, and policy and procedures of other agencies involved in regulatory

veterinary medicine. (see Target Animal Safety Guidelines for New Animal 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 dependent 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-observed-effect-level (NOEL), but to provide assurance that a adequate margin 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 following a dermal exposure to a single dose of a test substance or to repeat

applications to achieve dose. For purposes of domestic animal safety studies, the test substance is always the final formulation of a pesticide product.

Adverse effect is an undesirable effect reflected in the animal by alterations 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. milligrams per kilogram).

Margin of safety is the difference between the effective dose (recommended 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 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 (3× and 5× the recommended 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 effects 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 (1×). For exaggerated doses (3× and 5× the recommended dose), products specifically prepared for this type of study that

contain higher concentrations (3× and 5×) of the active ingredient are preferred. See additional discussion under paragraph (g)(3)(iii) of this guideline.

(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 5× the recommended 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 representative 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 product 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 acclimated 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 recommended. 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 0×, 1×, 3×, and 5× the recommended

dose. The targeted adequate margin of safety is 5×. Consideration will

be given to products with less than a 5× 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 5× level.

The vehicle should contain the inert ingredients at the maximum levels

that would appear in the 5× formulation.

(iii) Methods of achieving exaggerated doses. It is preferred that

formulations be revised to include exaggerated amounts of the active ingredient. However, if this cannot be achieved because of volume constraints

or the physical properties of the ingredients (active or inert), multiple treatments at frequent intervals during the same dosing period will be acceptable.

(A) Spray, dip or shampoo formulations may be applied at the recommended dose at hourly intervals to achieve an exaggerated dose, i.e.

three times for a 3× dose and five times for a 5× 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 observation 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 preparation 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 treatments 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 product.

(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 5× 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 observations.

(A) Ocular: Corneal opacities, nystagmus, pupillary changes,

blepharospasm, 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, comatose, depressed, sedated, restless, panting, convulsions, and aggression.

(E) Integument: Alopecia, haircoat condition, status of hydration, pruritus, and erythema.
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(F) Gastrointestinal: Consistency of stools, propulsive diarrhea, hyperactive 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 acclimation period, with the second measurement being taken immediately

prior to the initiation of treatment, and on day–7 and day–14 of the observation period.

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

(vii) The time of death should be reported for animals dying or sacrificed 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, hematocrit,mean corpuscular hemoglobin, red blood cell count,mean corpuscular

hemoglobin concentration, white blood cell count, white blood cell differential count, prothrombin time, and activated partial thromboplastic

time.

(B) Clinical chemistry. Glucose, creatinine, sodium, chloride, potassium, phosphorus, total protein, albumin, globulin, calcium, blood urea nitrogen, 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 consumption, 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 statistical 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 mortality 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 additional 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 D.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 Medicine, Food and Drug Administration, June 1989.