United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–250

June 1996

Health Effects Test

Guidelines

OPPTS 870.8223

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 870.8223. 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 795.223 Pharmacokinetic

Test.

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

bioavailability of a test substance after dermal administration, whether or

not the biotransformation of the test substance is qualitatively and quantitatively the same after dermal and oral administration, 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 definitions also apply to this test guideline.

Bioavailability refers to the rate and extent to which the administered

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

Relative percent of percutaneous absorption is defined as 100× the

ratio between total urinary excretion of compound following topical administration and total urinary excretion of compound following intravenous

injection.

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

utilized for investigating the test substance should be the rat, a species

for which historical data on the toxicity and carcinogenicity of several

compounds are available and which is used extensively in percutaneous

absorption studies.

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

should be 7 to 9 weeks old and weigh 125 to 175 g. Prior to testing the

animals should be selected at random for each group. Animals showing

signs of ill health should not be used.

(iii) Animal care. (A) The 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 25 ± 2 °C and humidity of

50 ± 10 percent with a 12–h light/dark cycle per day. The rats should be

kept in a quarantine facility for at least 7 days prior to use.

(B) During the acclimatization period, the rats should be housed in

cages on hardwood chip bedding. All animals should be provided with

conventional laboratory diets and water ad libitum.
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(2) Administration of test substance—(i) Test compound. Test

studies require the use of both nonradioactive test substance and 14C-labeled test substance. Both preparations are needed to investigate under

paragraph (b)(2) of this guideline. The use 14C-test substance is required

to investigate under paragraphs (b)(1), (b)(2), and (b)(3) of this guideline

because it will facilitate the work, improve the reliability of quantitative

determinations, and increase the probability of observing the presence of

previously unidentified metabolities.

(ii) Dosage and treatment. (A) Two doses should be used in the

study, a low dose and a high dose. When administered orally, the high

dose level should ideally induce some overt toxicity such as weight loss.

The low dose level should correspond to a no-effect level.

(B) The same high and low doses should be administered orally and

dermally.

(C) Oral dosing should be performed by gavage or by administering

encapsulated test substance.

(D) For dermal treatment, the doses should be applied at a volume

adequate to deliver the prescribed doses. The backs of the rats should be

lightly shaved with an electric clipper shortly before treatment. The dose

should be applied with a micropipet on 2 cm2 of the freshly shaven skin.

The dosed areas should be occluded with an aluminum foil patch which

is secured in place with adhesive tape.

(iii) Bioavailability study in rats. At least eight rats should receive

a single intravenous (low) dose of 14C-test substance and serial samples

of blood removed from four animals at 15 min, 30 min, 1 h, 8 h, 24

h, 48 h, and 96 h. All animals should be housed in metabolism cages

and urine and feces collected at 8, 24, 48, 72, and 96 h. The procedure

should be repeated with eight rats in which 14C-test substance is maintained in contact with the skin for the duration of the study (96 h). If

dermal absorption cannot be demonstrated, the study should be repeated

using a higher dose. Total radioactivity should be measured in the blood,

urine, and feces samples collected from all animals. The results should

be used to construct a blood concentration-time curve and to calculate

bioavailability by the ratio of the total 96-h urinary excretion of radioactivity after dermal and intravenous administration. Bioavailability is expressed as (percent dose dermal/percent dose intravenous) × 100 = percent

dermal absorption. Urine should be saved for metabolite identification, if

it becomes necessary.

(iv) Biotransformation in rats after oral and dermal administration. Eight rats should be dosed orally, and eight rats should be dosed

dermally (96-h contact) with the high dose of 14C-test substance. The results of the bioavailability study (see paragraph (d)(2)(iii) of this guideline)

should be evaluated first to ensure that the dermal dose applied will result
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in the appearance of radioactivity in the urine. All animals should be

housed in metabolism cages allowing for separate collection of urine and

feces at 8, 24, 48, 72, and 96 h. The parent compound and any metabolite

that comprises greater than 10 percent of the dose should be identified

in the urine. These results should be qualitatively compared to the urinary

excretion data obtained in the low dose bioavailability study (see paragraph

(d)(2)(iii) of this guideline); metabolites in the low dose urine should also

be identified if a different pattern of metabolism is evident.

(v) Repeated dosing study. Four rats should receive a series of single

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

14 days, followed at 24 h after the last dose by a single oral dose of

14C-test substance. Each dose should be at the low-dose level. If the pattern of urinary metabolite excretion is qualitatively different from that obtained with the orally dosed animals in the single-dose biotransformation

study at 24 and 48 h (see paragraph (d)(2)(iv) of this guideline),

metabolites should be identified in accordance with the procedure given

in paragraph (d)(2)(iii) of this guideline.

(vi) Skin washing study. If greater than 10 percent of test substance

is absorbed through the skin (see paragraphs (d)(2)(ii) and (d)(2)(iii) of

this guideline) then a washing efficacy experiment should be performed

to assess the extent of removal of the applied test substance by washing

with soap and water. Four rats should be lightly anesthetized and treated

with a dermal dose of test compound previously shown to result in measurable percutaneous absorption greater than 10 percent. Soon after application (5 to 10 min) the treated animals should be washed with soap and

water, then housed in individual metabolism cages for excreta collection.

Measurements of total radioactivity in urine and feces should be made

in the same manner as described in paragraph (d)(2)(iii) of this guideline.

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

presented in tabular form.

(2) Evaluation of results. All observed results, quantitative or incidental, 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) Species, strain, and supplier of laboratory animals.

(ii) Information on the degree (i.e., specific activity for a radiolabel)

and sites of labeling of the test substances.

(iii) A full description of the sensitivity and precision of all procedures used to produce the data.
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(iv) Relative percent absorption by the dermal route for rats administered low and high doses of 14C-test substance, compared with 100 percent

of the intravenous dose.

(v) Quantity of isotope, together with percent recovery of the administered dose, in feces, urine, and blood.

(vi) Biotransformation pathways and quantities of the test substance

and metabolites in urine collected after administering single high and low

oral and dermal doses.

(vii) Biotransformation pathways and quantities of test substance and

metabolites in urine collected after administering repeated low doses of

test substance to rats.