United States Prevention, Pesticides EPA712-C-96-250
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 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 870.8223. Pharmacokinetic test.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing 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 har-
monized 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 quan-
titatively 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 lOOx the
ratio between total urinary excretion of compound following topical ad-
ministration 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 environ-
mentally 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-la-
beled 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)(l), (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 main-
tained 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 radioactiv-
ity after dermal and intravenous administration. Bioavailability is ex-
pressed as (percent dose dermal/percent dose intravenous) x 100 = percent
dermal absorption. Urine should be saved for metabolite identification, if
it becomes necessary.
(iv) Biotransformation in rats after oral and dermal administra-
tion. 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 re-
sults 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 pat-
tern of urinary metabolite excretion is qualitatively different from that ob-
tained 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 measur-
able percutaneous absorption greater than 10 percent. Soon after applica-
tion (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 inci-
dental, 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 proce-
dures used to produce the data.
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(iv) Relative percent absorption by the dermal route for rats adminis-
tered 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 adminis-
tered 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.
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