United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–254

June 1996

Health Effects Test

Guidelines

OPPTS 870.8340

Oral and Inhalation

Pharmacokinetic Test

‘‘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 870.8340 Oral and inhalation pharmacokinetic test.

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances

Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this harmonized OPPTS test guideline is OPPT 40 CFR 795.230 Oral and Inhalation Pharmacokinetic Test.

(b) Purpose. The purpose of these studies is to determine

bioavailability of test substance after oral and inhalation exposure; whether

or not the biotransformation of the test substance is qualitatively and quantitatively the same after oral and inhalation exposure; and whether or not

the biotransformation of the test substance is changed qualitatively or

quantitatively by repeated dosing.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR

Part 792—Good Laboratory Practice Standards (GLP) apply to this test

guideline. The following definition also applies to this test guideline.

Bioavailability refers to the rate and extent to which an administered

chemical substance compound is absorbed, i.e. reaches the systemic circulation.

(d) Test procedures—(1) Animal selection—(i) Species. The rat

should be used for pharmacokinetics testing because it has been used extensively for metabolic and toxicological studies.

(ii) Animals. Adult male and female Fischer 344 rats should be used.

The rats should be 7 to 9 weeks old. Prior to testing, the animals are

selected at random for each group. Animals showing signs of ill health

should not be used.

(iii) Animal care. Animals should be housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms should be

maintained at a temperature of 24 ± 2 °C and humidity 50 ± 10 percent

with a 12-h light/dark cycle per day. The rats should be isolated for at

least 7 days prior to use, and their health status should then be evaluated.

The animals should be acclimated to the experimental environment for

a minimum of 48 h prior to treatment. Certified feed and water should

be provided ad libitum.

(iv) Numbers. (A) At least eight animals (four males and four females) should be used at each dose level.

(B) Females should be nulliparous and nonpregnant.

(2) Administration of the test substance—(i) Test substance. The

test substance should be at least 99 percent pure. The studies require the
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use of both nonradioactive and 14C-labeled test substance. Both preparations are needed to investigate the provisions of paragraph (b)(2) of this

guideline. The use of 14C-test substance is recommended for the provisions

in paragraphs (b)(1), (b)(2), and (b)(3) of this guideline in order to facilitate the work, improve the reliability of quantitative determinations, and

increase the probability of observing previously unidentified metabolites.

(ii) Dosage and treatment—(A) Oral study. At least two doses

should be used in the study, a low and a high dose. When administered

orally, the high dose should induce some overt toxicity such as weight

loss. The low dose should not induce observable effects attributable to

the test substance. Oral dosing should be performed by gavage using an

appropriate vehicle.

(B) Inhalation study. Three concentrations should be used in the

study. Upon exposure, the two higher concentrations should ideally induce

some overt symptoms of toxicity, although the intermediate concentration

may be excluded from this condition. The lowest concentration should not

induce observable effects attributable to the test substance.

(iii) Determination of bioavailability—(A) Oral studies. (1) Group

A (a minimum of eight animals, four males and four females) should be

dosed once orally with the low dose of 14C-labeled test substance.

(2) Group B (a minimum of eight animals, four males and four females) should be dosed once orally with the high dose of 14C-labeled test

substance.

(B) Inhalation studies. (1) Group C (a minimum of eight animals,

four males and four females) should be exposed (6 h) to a mixture of

nonradioactive test substance in air at the prescribed low test substance

concentration.

(2) Group D (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of nonradioactive test substance in air at the prescribed intermediate test substance concentration.

(3) Group E (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of nonradioactive test substance in air at the prescribed high concentration.

(4) Group F (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14C-labeled test substance

in air at the prescribed low test substance concentration.

(5) Group G (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14C-labeled test substance

in air at the prescribed intermediate test substance concentration.
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(6) Group H (a minimum of eight animals, four males and four females) should be exposed (6 h) to a mixture of 14C-labeled test substance

in air at the prescribed high test substance concentration.

(C) Collection of excreta. After oral administration (Groups A and

B) and inhalation exposure (Groups F through H), the rats should be

placed in individual metabolic cages and excreta (urine, feces, and expired

air) should be collected from 0 to 24 h and then from 24 to 48 h

posttreatment, or until 90 percent of the dose has been excreted, whichever

occurs first.

(D) Kinetic studies. Groups C through E should be used to determine

the concentration of the test substance in blood at 0, 5, 10, 15, and 30

min, and at 1, 2, 4, 8, 16, 24, and 48 h after initiation of inhalation exposure.

(E) Repeated dosing study. Rats (a minimum of eight animals, four

males and four females) should receive a series of single daily oral doses

of nonradioactive test substance over a period of at least 7 days, followed

at 24 h after the last dose by a single oral dose of 14C-labeled test substance. Each dose should be at the low-dose level. Urine should be collected from 0 to 24 h and then 24 to 48 h after administering the 14Clabeled test substance.

(3) Observation of animals—(i) Bioavailability—(A) Blood levels.

The levels of total 14C-label should be determined in whole blood, blood

plasma, or blood serum of each rat at 0, 4, 8, 16, 24, and 48 h after

dosing rats in Groups A–B and F–H.

(B) Expired air, urinary and fecal excretion. The quantities of total

14C-label eliminated in expired air, urine, and feces by each rat in Groups

A and B and F through H should be determined in collections made from

0 to 24 h and then 24 to 48 h after dosing and, if necessary, daily thereafter

until at least 90 percent of the dose has been excreted or until 7 days

after dosing, whichever occurs first.

(C) Tissue distribution. The concentration and quantity of 14C-label

in tissue and organs should be determined at the time of sacrifice for each

rat in Groups A and B, F through H, and the repeated-dosing group.

(ii) Biotransformation after oral and inhalation exposure. Appropriate qualitative and quantitative methods should be used to assay urine

specimens collected from each rat in Groups A and B and F through H.

Suitable enzymatic steps should be used to distinguish, characterize, and

quantify conjugated and unconjugated metabolites of the test substance.

(iii) Changes in biotransformation. Appropriate qualitative and

quantitative assay methodologies should be used to compare the composition of 14C-labeled components of urine collected from 0 to 24 h and
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then from 24 to 48 h after dosing rat Group A with those components

in the urine collected over the same intervals after administering the radioactive dose in the repeated dosing study.

(e) Data and reporting—(1) Treatment of results. Data should be

summarized in tabular form.

(2) Evaluation of results. All observed results should be evaluated

by an appropriate statistical method.

(3) Test report. In addition to the reporting requirements as specified

in 40 CFR part 792, subpart J, the following specific information should

be reported:

(i) Labeling site of the test substance.

(ii) A full description of the sensitivity and precision of all procedures

used to produce the data.

(iii) Quantity of isotope, together with percent recovery of the administered dose in feces, urine, expired air, and blood for both routes of administration.

(iv) Quantity and distribution of 14C-test substance in bone, brain,

fat, gonads, heart, kidney, liver, lung, muscle, spleen, tissue which displayed pathology, and residual carcass.

(v) Biotransformation pathways and quantities of test substance and

its metabolites in urine, feces, and expired air collected after oral administration (single low and high doses) and inhalation exposure (low, intermediate, and high concentrations).

(vi) Biotransformation pathways and quantities of the test substance

and its metabolites in urine collected after repeated administration of test

substance to rats.

(vii) Pharmacokinetic models, if any, developed from the experimental data.

(4) Counting efficiency. Data should be made available to the Agency upon request.