United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–255

June 1996

Health Effects Test

Guidelines

OPPTS 870.8360

Pharmacokinetics of

Isopropanal

‘‘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 870.8360 Pharmacokinetics of isopropanal.

(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 795.231 Pharmacokinetics

of Isopropanal.

(b) Purpose. The purpose of these studies is to ascertain whether the

pharmacokinetics and metabolism of the test substance are similar after

oral and inhalation administration; determine bioavailability of the test substance after oral and inhalation administration; and to examine the effects

of repeated dosing on the pharmacokinetics and metabolism of the test

substance.

(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 definitions also apply to this test guideline.

Bioavailability refers to the rate and relative amount of administered

test substance which reaches the systemic circulation.

Metabolism means the study of the sum of the processes by which

a particular substance is handled in the body, and includes absorption, tissue distribution, biotransformation, and excretion.

Pharmacokinetics means the study of the rates of absorption, tissue

distribution, biotransformation, and excretion.

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

should be used because it has been used extensively for metabolic and

toxicological studies.

(ii) Test animals. For pharmacokinetics testing, adult male and female rats (Fischer 344 or strain used for major toxicity testing), 7 to 9

weeks of age, should be used. The animals should be purchased from a

reputable dealer and should be identified upon arrival at the testing laboratory. The animals should be selected at random for the testing groups and

any animal showing signs of ill health should not be used. In all studies,

unless otherwise specified, each test group should contain at least four

animals of each sex for a total of at least eight animals.

(iii) Animal care. (A) Animal care and housing should be in accordance with DHEW Publication No. (NIH)–85–23 (1985), Guidelines for the

Care and Use of Laboratory Animals.

(B) The animals should be housed in environmentally controlled

rooms with at least 10 air changes per hour. The rooms should be main-
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tained at a temperature of 22 ± 2 °C and humidity of 50 ± 20 percent with

a 12–h light/dark cycle per day. The animals should be kept in a quarantine

facility for at least 7 days prior to use and should be acclimated to the

experimental environment for a minimum of 48 h prior to treatment.

(C) During the acclimatization period, the animals should be housed

in suitable cages. All animals should be provided with certified feed and

tap water ad libitum.

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

of radioactive test substance is required for all materials balance and

metabolite identification requirements of the study. The purity of both radioactive and nonradioactive test substance should be greater than 99 percent. The radioactive and nonradioactive substances should be

chromatographed separately and together to establish purity and identity.

If the purity is less than 99 percent or if the chromatograms differ significantly, EPA should be consulted.

(ii) Dosage and treatment—(A) Intravenous. The low dose of test

substance, in an appropriate vehicle, should be administered intravenously

to four rats of each sex.

(B) Oral. Two doses of test substance should be used in the oral

portion of the study, a low dose and a high dose. The high dose should

induce some overt toxicity, such as weight loss. The low dose level should

correspond to a no-observed-effect level. The oral dosing should be accomplished by gavage or by administering an encapsulated test substance.

If feasible, the same high and low doses should be used for oral and dermal

studies.

(C) Inhalation. Two concentrations of the test substance should be

used in this portion of the study, a low concentration and a high concentration. The high concentration should induce some overt toxicity, while the

low concentration should correspond to a no-observed-effect level. Inhalation treatment should be conducted using a nose-cone or head-only apparatus to prevent ingestion of the test substance through grooming.

(iii) Dosing and sampling schedule. After administration of the test

substance, each rat should be placed in a separate metabolic unit to facilitate collection of excreta. For the inhalation studies, excreta from the rats

should also be collected during the exposure periods. At the end of each

collection period, the metabolic units should be cleaned to recover any

excreta that might adhere to the cages. All studies, except the repeated

dose study, should be terminated at 7 days, or after at least 90 percent

of the radioactivity has been recovered in the excreta, whichever occurs

first.

(A) Intravenous study. Group A should be dosed once

intravenousely at the low dose of test substance.
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(B) Oral studies. (1) Group B should be dosed once orally with the

low dose of the test substance.

(2) Group C should be dosed once orally with the high dose of the

test substance.

(C) Inhalation studies. A single 6–h exposure period should be used

for each group.

(1) Group D should be exposed to a mixture of the test substance

in air at the low concentration.

(2) Group E should be exposed to a mixture of test substance in air

at the high concentration.

(D) Repeated dosing study. Group F should receive a series of single

daily oral low doses of nonradioactive test substance over a period of at

least 7 consecutive days. A single oral low dose of radioactive test substance should be administered 24 h after the last nonradioactive dose. Following dosing with radioactive substance, the rats should be placed in individual metabolic units as described in paragraph (d)(2)(iii) of this guideline. The study should be terminated 7 days after the last dose, or after

at least 90 percent of the radioactivity has been recovered in the excreta,

whichever occurs first.

(3) Types of studies—(i) Pharmacokinetics studies. Groups A

through F should be used to determine the kinetics of absorption of the

test substance. In groups administered the substance by intravenous or oral

routes, (i.e., Groups A, B, C, F), the concentration of radioactivity in blood

and excreta including expired air should be measured following administration. In groups administered the substance by the inhalation route (i.e.,

Groups D and E), the concentration of radioactivity in blood should be

measured at selected time intervals during and following the exposure period. In the groups administered the substance by inhalation (i.e., Groups

D and E), the concentration of radioactivity in excreta (including expired

air) should be measured at selected time intervals following the exposure

period. In addition, in the groups administered the substance by inhalation,

the concentration of test substance in inspired air should be measured at

selected time intervals during the exposure period.

(ii) Metabolism studies. Groups A through F should be used to determine the metabolism of the test substance. Excreta (urine, feces, and expired air) should be collected for identification and quantification of test

substance and metabolites.

(4) Measurements—pharmacokinetics. Four animals from each

group should be used for these measurements.

(i) Bioavailability. The levels of radioactivity should be determined

in whole blood, blood plasma or blood serum at 15 min, 30 min, 1, 2,
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3, 6, 9, and 18 h after dosing; and at 30 min, 3, 6, 6.5, 7, 8, 9, 12, and

18 h after initation of inhalation exposure.

(ii) Extent of absorption. The total quantities of radioactivity should

be determined for excreta collected daily for 7 days, or after at least 90

percent of the radioactivity has been recovered in the excreta, whichever

occurs first.

(iii) Excretion. The quantities of radioactivity eliminated in the urine,

feces, and expired air should be determined separately at appropriate time

intervals. The collection of the intact test substance or its metabolites, including carbon dioxide, may be discontinued when less than 1 percent

of the administered dose is found to be exhaled as radioactive carbon dioxide in 24 h.

(iv) Tissue distribution. At the termination of each study, the quantities of radioactivity in blood and in various tissues, including bone, brain,

fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin,

spleen, and residual carcass of each animal should be determined.

(v) Changes in pharmacokinetics. Results of pharmacokinetics

measurements (i.e., biotransformation, extent of absorption, tissue distribution, and excretion) obtained in rats receiving the single low oral dose

of test substance (Group B) should be compared to the corresponding results obtained in rats receiving repeated oral doses of test substance (Group

F).

(vi) Biotransformation. Appropriate qualitative and quantitative

methods should be used to assay urine, feces, and expired air collected

from rats. Efforts should be made to identify any metabolite which comprises 5 percent or more of the dose eliminated.

(vii) Changes in biotransformation. Appropriate qualitative and

quantitative assay methodology should be used to compare the composition

of radioactive substances in excreta from the rats receiving a single oral

dose (Groups B and C) with those in the excreta from rats receiving repeated oral doses (Group F).

(e) Data and reporting. The final test report should include the following:

(1) Presentation of results. Numerical data should be presented in

tabular form. Pharmacokinetics data should also be presented in graphical

form. Qualitative observations should be reported.

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

by an appropriate statistical method.
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(3) Reporting results. In addition to the reporting requirements as

specified in 40 CFR 792.185, the following specific information should

be reported:

(i) Species and strains of laboratory animals.

(ii) Chemical characterization of the test substance, including:

(A) For the radioactive test substance, information on the sites and

degree of radiolabeling, including type of label, specific activity, chemical

purity, and radiochemical purity.

(B) For the nonradioactive substance, information on chemical purity.

(C) Results of chromatography.

(iii) A full description of the sensitivity, precision, and accuracy of

all procedures used to generate the data.

(iv) Extent of absorption of the test substance as indicated by percent

absorption of the administered oral dose and total body burden after inhalation exposure.

(v) Quantity and percent recovery of radioactivity in feces, urine, expired air, and blood.

(vi) Tissue distribution reported as quantity of radioactivity in blood

and in various tissues, including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney, liver, lung, muscle, skin, spleen and in residual carcass

of each rat.

(vii) Biotransformation pathways and quantities of the test substance

and metabolites in excreta collected after administering single high and

low doses to rats.

(viii) Biotransformation pathways and quantities of the test substance

and metabolites in excreta collected after administering repeated low doses

to rats.

(ix) Pharmacokinetics models developed from the experimental data.