United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-253
June 1996
&EPA Health Effects Test
Guidelines
OPPTS 870.8320
Oral/Dermal
Pharmacokinetics
I
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
(7 U.S.C. 136, 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 870.8320 Oral/dermal pharmacokinetics.
(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 795.228 Oral/Dermal
Pharmac okinetic s.
(b) Purpose. The purpose of these studies is to ascertain whether the
pharmacokinetics and metabolism of a chemical substance or mixture
("test substance") are similar after oral and dermal administration; deter-
mine bioavailability of a test substance after oral and dermal administra-
tion; 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, tis-
sue distribution, biotransformation, and excretion.
Percent absorption means lOOx the ratio between total excretion of
radioactivity following oral or dermal administration and total excretion
following intravenous administration of test substance.
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 for pharmacokinetics testing because it has been used ex-
tensively for metabolic and toxicological studies. For dermal
bioavailability studies, the rat and the miniature pig should be used.
(ii) Test animals. For pharmacokinetics testing and dermal studies,
adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, should
be used. For dermal studies, young adult miniature pigs should also 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 test 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.
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(iii) Animal care. (A) The animals should be housed in environ-
mentally controlled rooms with at least 10 air changes per hour. The rooms
should be maintained at a temperature of 24 ±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 accli-
mated to the experimental environment for a minimum of 48 h prior to
administration of the test substance.
(B) 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. The miniature pig diet should be supplemented with
adequate amounts of ascorbic acid in the drinking water.
(2) Administration of test substance—(i) Test substance. The use
of a radioactive test substance is required for all studies. The purity, radio-
active and nonradioactive, should be greater than 99 percent. The radio-
active and nonradioactive test substances should be chromatographed sepa-
rately 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 groups of rats and miniature pigs of each sex. If feasible, the same
low dose should be used for intravenous, oral, and dermal studies.
(B) Oral. Two doses of text substance should be used in the oral
study, a low dose, and a high dose. The high dose should induce some
overt toxicity, such as weight loss. The low dose should correspond to
a no-observed-effect level. The oral dosing should be accomplished by
gavage or by administering the encapsulated test substance. If feasible,
the same high and low doses should be used for oral and dermal studies.
(C) Dermal—(7) Dermal treatment. For dermal treatment, two
doses, comparable to the low and high oral doses, should be dissolved
in a suitable vehicle and applied in volumes adequate to deliver com-
parable doses. The backs of the animals should be lightly shaved with
an electric clipper 24 h before treatment. The test substance should be
applied to the intact shaven skin (approximately 2 cm2 for rats and 5 cm2
for miniature pigs). The dosed areas should be protected with a suitable
porous covering which is secured in place, and the animals should be
housed separately.
(2) Washing efficacy study. Before initiation of the dermal absorp-
tion studies, an initial washing efficacy experiment should be conducted
to assess the removal of the applied low dose of the test substance by
washing the exposed skin area with soap and water and an appropriate
organic solvent. The low dose should be applied to four rats and four
miniature pigs in accordance with paragraph (d)(2)(ii)(C)(7) of this guide-
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line. After application (5 to 10 min), the treated areas of two rats and
two miniature pigs should be washed with soap and water and the treated
areas of the remaining rats and pigs should be washed with an appropriate
solvent. The amounts of test substance recovered in the washings should
be determined to assess efficacy of its removal by washing.
(iii) Dosing and sampling schedule—(A) Rat studies. After admin-
istration of the test substance, each rat should be placed in a metabolic
unit to facilitate collection of excreta. For the dermal studies, excreta from
the rats should also be collected during the 6-h exposure periods. At the
end of each collection period, the metabolic units should be cleaned to
recover any excreta that might adhere to them. All studies, except the re-
peated dosing 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.
(7) Intravenous study. Group A should be dosed once intravenously
at the low dose of test substance.
(2) Oral study, (z) Group B should be dosed once orally with the
low dose of test substance.
(zz) Group C should be dosed once orally with the high dose of test
substance.
(3) Dermal studies. Unless precluded by corrosivity, the test sub-
stance should be applied and kept on the skin for a minimum of 6 h.
At the time of removal of the porous covering, the treated area should
be washed with an appropriate solvent to remove any test substance that
may be on the skin surface. Both the covering and the washing should
be assayed to recover residual radioactivity. At the termination of the stud-
ies, each animal should be sacrificed and the exposed skin area removed.
An appropriate section of the skin should be solubilized and assayed for
radioactivity to ascertain if the skin acts as a reservoir for the test sub-
stance. Studies on the dermal absorption of corrosive test substances
should be discussed with EPA prior to initiation.
(z) Group D should be dosed once dermally with the low dose of
test compound.
(zz) Group E should be dosed once dermally with the high dose of
the test substance.
(4) 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 days. At 24 h after the last nonradioactive dose, a single oral low
dose of radioactive test substance should be administered. Following dos-
ing with the radioactive substance, the rats should be placed in individual
metabolic units as described in paragraph (d)(2)(iii) of this guideline. The
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study should be terminated at 7 days after the last dose, or after at least
90 percent of the radioactivity has been recovered in the excreta, which-
ever occurs first.
(B) Miniature pig studies. For all miniature pig studies, the test
groups should consist of four young adult animals. After administration
of the test substance, each miniature pig should be kept in a metabolic
unit to facilitate collection of excreta. At the end of each collection period,
the metabolic units are to be cleaned to recover any excreta that might
adhere to them. All studies should be terminated at 7 days, or after at
least 90 percent of the radioactivity has been recovered in the excreta,
whichever occurs first.
(7) Intravenous study. Group G is to be dosed once intravenously
at the low dose of the test substance.
(2) Dermal studies. Following the experimental guidance described
in paragraph (d)(2)(iii)(A)(3) of this guideline:
(z) Group H should be dosed once dermally with the low dose of
test substance.
(zz) Group I should be dosed once dermally with the high dose of
the test substance.
(3) Types of studies—(i) Pharmacokinetics studies—(A) Rat stud-
ies. Groups A through F should be used to determine the kinetics of ab-
sorption of the test substance. In the group administered the test substance
by intravenous routes, (i.e., Group A), the concentration of radioactivity
in blood and excreta should be measured following administration. In
groups administered the test substance by the oral and dermal route (i.e.,
Groups B, C, D, E, and F), the concentration of radioactivity in blood
and excreta should be measured at selected time intervals during and fol-
lowing the exposure period.
(B) Miniature pig studies. Groups G, H, and I should be used to
determine the extent of dermal absorption of the test substance. The
amount of radioactivity in excreta should be determined at selected time
intervals.
(ii) Metabolism studies—Rat studies. Groups A through F should
be used to determine the metabolism of the test substance. Urine, feces,
and expired air should be collected for identification and quantification
of the test substance and metabolites.
(4) Measurements—(i) Pharmacokinetics. Four animals from each
group should be used for these purposes.
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(A) Rat studies—(7) Bioavailability. The levels of radioactivity
should be determined in whole blood, blood plasma or blood serum at
15 and 30 min and at 1, 2, 8, 24, 48, and 96 h after initiation of dosing.
(2) Extent of absorption. The total quantities of radioactivity should
be determined for excerta collected daily for 7 days or until at least 90
percent of the radioactivity has been recovered in the excreta.
(3) Excretion. The quantities of radioactivity eliminated in the urine,
feces, and expired air should be determined separately at appropriate time
intervals. The collection of carbon dioxide may be discontinued when less
than one percent of the dose is found to be exhaled as radioactive carbon
dioxide in 24 h.
(4) Tissue distribution. At the termination of each study, the quan-
tities of radioactivity in blood and in various tissues, including bone, brain,
fat, gastrointestinal tract, gonads, heart, kidney, liver, lungs, muscle, skin,
and residual carcass of each animal should be determined.
(5) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., bioavailability and extent of absorption, tissue distribu-
tion, and excretion) obtained in rats receiving the single low oral dose
of the test substance (Groups B and C) should be compared to the cor-
responding results obtained in rats receiving repeated oral doses of the
test substance (Group F).
(B) Miniature pig studies—Extent of absorption. The total quan-
tities of radioactivity should be determined for excreta daily for 7 days
or until at least 90 percent of the test substance has been excreted.
(ii) Metabolism. Four animals from each group should be used for
these purposes.
(A) Rat studies—(7) 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 administered dose and the major
radioactive components of blood.
(2) Changes in biotransformation. Appropriate qualitative and quan-
titative assay methodology should be used to compare the composition
of radioactive compounds in excreta from rats receiving a single oral dose
(Groups B and C) with those in the excreta from rats receiving repeated
oral doses (Group H).
(e) Data and reporting. The final test report should include the fol-
lowing:
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(1) Presentation of results. Numerical data should be presented in
tabular form. Pharmacokinetic data should also be presented in graphical
form. Qualitative observations should also be reported.
(2) Evaluation of results. All quantitative results should be evaluated
by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, subpart J, 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 substances, information on the site(s) and
degree of radiolabeling, including type of label, specific activity, chemical
purity, and radiochemical purity.
(B) For the nonradioactive compound, information on chemical pu-
rity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy of
all procedures used to generate the data.
(iv) Percent absorption of test substance after oral and dermal expo-
sures to rats and dermal exposure to miniature pigs.
(v) Quantity and percent recovery of radioactivity in feces, urine, ex-
pired air, and blood. In dermal studies on rats and miniature pigs, include
recovery data for skin, skin washings, and residual radioactivity in the
covering as well as results of the washing efficacy study.
(vi) Tissue distribution reported as quantity of radioactivity in blood
and in various tissues, including bone, brain, fat, gastrointestinal tract, go-
nads, heart, kidney, liver, lung, muscle, skin and in residual carcass of
rats.
(vii) Materials balance developed from each study involving the assay
of body tissues and excreta.
(viii) Biotransformation pathways and quantities of test substance and
metabolites in excreta collected after administering single high and low
doses to rats.
(ix) Biotransformation pathways and quantities of the test substance
and metabolites in excreta collected after administering repeated low doses
to rats.
(x) Pharmacokinetics models developed from the experimental data.
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