United States Prevention, Pesticides EPA712-C-96-254
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 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 870.8340 Oral and inhalation 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 795.230 Oral and Inhala-
tion 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 quan-
titatively 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 cir-
culation.
(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.
(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 con-
trolled 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 fe-
males) 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 prepara-
tions 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)(l), (b)(2), and (b)(3) of this guideline in order to facili-
tate 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. (7) 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 fe-
males) should be dosed once orally with the high dose of 14C-labeled test
substance.
(B) Inhalation studies. (7) Group C (a minimum of eight animals,
four males and four females) should be exposed (6 h) to a mixture of
nonradio active test substance in air at the prescribed low test substance
concentration.
(2) Group D (a minimum of eight animals, four males and four fe-
males) should be exposed (6 h) to a mixture of nonradioactive test sub-
stance in air at the prescribed intermediate test substance concentration.
(3) Group E (a minimum of eight animals, four males and four fe-
males) should be exposed (6 h) to a mixture of nonradioactive test sub-
stance in air at the prescribed high concentration.
(4) Group F (a minimum of eight animals, four males and four fe-
males) 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 fe-
males) 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 fe-
males) 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 expo-
sure.
(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 sub-
stance. Each dose should be at the low-dose level. Urine should be col-
lected from 0 to 24 h and then 24 to 48 h after administering the 14C-
labeled 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. Appro-
priate 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 composi-
tion 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 radio-
active 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 admin-
istered dose in feces, urine, expired air, and blood for both routes of ad-
ministration.
(iv) Quantity and distribution of 14C-test substance in bone, brain,
fat, gonads, heart, kidney, liver, lung, muscle, spleen, tissue which dis-
played pathology, and residual carcass.
(v) Biotransformation pathways and quantities of test substance and
its metabolites in urine, feces, and expired air collected after oral adminis-
tration (single low and high doses) and inhalation exposure (low, inter-
mediate, 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 experi-
mental data.
(4) Counting efficiency. Data should be made available to the Agen-
cy upon request.
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