United States Prevention, Pesticides EPA712-C-96-255
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 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 870.8360 Pharmacokinetics of isopropanal.
(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 795.231 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 sub-
stance 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, tis-
sue 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 fe-
male 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 labora-
tory. 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 accord-
ance 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 ra-
dioactive and nonradioactive test substance should be greater than 99 per-
cent. 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 signifi-
cantly, 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 ac-
complished 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 concentra-
tion. The high concentration should induce some overt toxicity, while the
low concentration should correspond to a no-observed-effect level. Inhala-
tion treatment should be conducted using a nose-cone or head-only appara-
tus 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 facili-
tate 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. (7) 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.
(7) 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 sub-
stance should be administered 24 h after the last nonradioactive dose. Fol-
lowing dosing with radioactive substance, the rats should be placed in indi-
vidual metabolic units as described in paragraph (d)(2)(iii) of this guide-
line. 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 adminis-
tration. 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 pe-
riod. 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 deter-
mine the metabolism of the test substance. Excreta (urine, feces, and ex-
pired 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, in-
cluding carbon dioxide, may be discontinued when less than 1 percent
of the administered dose is found to be exhaled as radioactive carbon diox-
ide in 24 h.
(iv) 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,
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 distribu-
tion, and excretion) obtained in rats receiving the single low oral dose
of test substance (Group B) should be compared to the corresponding re-
sults 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 com-
prises 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 re-
peated oral doses (Group F).
(e) Data and reporting. The final test report should include the fol-
lowing:
(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 inhala-
tion exposure.
(v) Quantity and percent recovery of radioactivity in feces, urine, ex-
pired air, and blood.
(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, 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.
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