United States Prevention, Pesticides EPA712-C-96-256
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8380
Inhalation and Dermal
Pharmacokinetics of
Commercial Hexane
'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 870.8380 Inhalation and dermal pharmacokinetics of com-
mercial hexane.
(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 795.232 Inhalation and
Dermal Pharmacokinetics of Commercial Hexane.
(b) Purpose. The purpose of these studies is to determine the
bioavailability of the test substances after dermal and inhalation adminis-
tration; to compare the pharmacokinetics and metabolism of the test sub-
stances after intravenous, dermal, and inhalation administration; and to ex-
amine the effects of repeated doses on the pharmacokinetics and metabo-
lism of the test substances.
(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 relative amount of administered test sub-
stance which reaches the systemic circulation and the rate at which this
process occurs.
High dose should not exceed the lower explosive limit (LEL) and
should induce minimal toxicity.
Low dose should correspond to one-tenth of the high dose.
Metabolism means the sum of the enzymatic and nonenzymatic proc-
esses by which a particular substance is handled in the body.
Pharmacokinetics means the study of the rates of absorption, tissue
distribution, biotransformation, and excretion.
Test substance refers to the unlabeled and both radiolabeled mixtures
(14C-w-hexane and 14C-methylcyclopentane (MCP)) of commercial hexane
used in the testing.
(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) Test animals. Adult male and female rats should be used for
testing. The rats should be 7 to 9 weeks old and their weight range should
be comparable from group to group. The animals should be purchased
from a reputable dealer and should be permanently identified upon arrival.
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The animals should be selected at random for the testing groups, and any
animal showing signs of ill health should not be used.
(iii) Animal care. (A) Animal care and housing should be in accord-
ance with DHHS/PHS NIH Publication No. 86-23 (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-
tained at a temperature of 18 to 26 °C and humidity of 40 to 70 percent
with a 12-h light/dark cycle per day. The animal subjects 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 rats should be housed in
suitable cages. All animals should be provided with certified feed and tap
water ad libitum.
(2) Administration of test substances—(i) Test substances. The
study will require the use of both radiolabeled and unlabeled test sub-
stances. All unlabeled commercial hexane should be from the same lot
number. Two kinds of radiolabeled test substances will be tested.
14C-w-hexane should be the only radiolabeled component of one, and
14C-MCP should be the only radiolabeled component of the other test sub-
stance. The use of both radiolabeled test substances is required for all
pharmacokinetics and metabolism studies described in this rule, except for
the bioavailability measurements required in paragraph (d)(4)(i)(A) of this
guideline. The bioavailability measurements need only be conducted with
the test substance containing 14C-w-hexane or an unlabeled test substance
may be used if it can be demonstrated that the analytical sensitivity of
the method used with the unlabeled test substance is equal to or greater
than the sensitivity which could be obtained with the radiolabeled test sub-
stance. If an unlabeled test substance is used for bioavailability measure-
ments, these measurements should be extended to include relevant
metabolites of w-hexane. These test substances should contain
at least 40 liquid volume percent but no more than 55 liquid volume per-
cent w-hexane and no less than 10 liquid volume percent
methylcyclopentane (MCP) and otherwise conform to the specifications
prescribed in the American Society for Testing and Materials Designation
D 1836-83 (ASTM D 1836), Standard Specification for Commercial
Hexanes.Copies of this material may be obtained from the American Soci-
ety for Testing and Materials (ASTM), 1916 Race Street, Philadelphia,
PA 19103.
(ii) Dosage and treatment—(A) Intravenous. An appropriate dose
of the test substance should be administered intravenously. The intra-
venous data obtained in this portion of the study should be suitable for
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the determination of absorption, distribution, and excretion parameters of
the test substance. Factors that should be considered in the selection of
the intravenous doses are: The acute toxicity of the test substance, the
availability of a suitable vehicle (if saline is unsuitable) and the solubility
of the test substance in the vehicle.
(B) Inhalation. Two concentrations of each test substance should be
used in this portion of the study, a low concentration and a high concentra-
tion. The high concentration should induce minimal toxicity, but should
not exceed the LEL. The low concentration should correspond to one-tenth
of the high concentration. Inhalation treatment should be conducted using
a nose-cone or head-only apparatus to reduce ingestion of the test sub-
stance through grooming or dermal absorption.
(C) Dermal. Dermal absorption studies should be conducted as de-
scribed under paragraph (f)(l) of this guideline or by some other suitable
method, care being taken because of the significant volatility of the test
substances. The high and low doses should be tested in rats.
(iii) Dosing and sampling schedule. Each experimental group should
contain at least four animals of each sex. After administration of the test
substance, each rat should be placed in an individual metabolic unit for
collection of urine, feces, and expired air. For the dermal 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 units. All studies, except
the repeated dose studies, should be terminated at 7 days, or after at least
90 percent of the administered radioactivity has been recovered in the ex-
creta, whichever occurs first. All studies described below should be con-
ducted separately with each radiolabeled test substance.
(A) Intravenous study. Group A should be given a single intra-
venous dose of the radiolabeled test substance to result in a level of com-
mercial hexane in the blood that approximates the level from the other
routes of exposure so that the data can be used to determine absorption
and excretion parameters.
(B) Inhalation studies. A single 6-h exposure period should be used
for each group.
(7) Group B should be exposed to a mixture of the radiolabeled test
substance in air at the low concentration.
(2) Group C should be exposed to a mixture of the radiolabeled test
substance in air at the high concentration.
(C) Dermal studies. The test substance should be applied and kept
on the skin for a minimum of 6 h. The covering apparatus components
should be assayed to recover residual radioactivity. At the termination of
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the studies, each animal should be sacrificed and the exposed skin area
removed. An appropriate section of the skin should be solubilized and as-
sayed for radioactivity to ascertain whether the skin acts as a reservoir
for the test substance.
(7) Group D should be given one dermal, low dose of the radiolabeled
test substance.
(2) Group E should be given one dermal, high dose of the
radiolabeled test substance.
(D) Repeated dosing study. Group F should receive a series of single
daily 6-h inhalation exposures to unlabeled test substance at the low dose
over a period of at least 7 days. A single 6-h inhalation exposure to the
radiolabeled test substance at the low dose should be administered 24 h
after the last unlabeled exposure. Following administration of the
radiolabeled substance, the rats should be placed in individual metabolic
units and excreta collected. The study should be terminated 7 days after
the last exposure, 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 animal subjects administered the test substance intra-
venously (i.e. Group A), the concentration of test substance in blood and
excreta should be measured following administration. In animal subjects
administered the test substance by the inhalation and dermal routes (i.e.
Groups B through F), the concentration of test substance in blood should
be measured at selected time intervals during and following the exposure
period. In animal subjects administered the test substance by the inhalation
route (i.e. Groups B, C, and F) the concentration of test substance in ex-
creta should be measured following exposure. In animal subjects adminis-
tered the test substance by the dermal route (i.e. Groups D and E) the
concentration of test substance in excreta should be measured during and
following exposure. These measurements allow calculation of uptake, half
lives, and clearance. In addition, in the groups administered the test sub-
stance by inhalation (i.e. Groups B, C, and F), the concentration of test
substance in the exposure chamber 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 measurement of the
quantities of test substance and metabolites.
(4) Measurements—(i) Pharmacokinetics. At least four animals
from each group should be used for these purposes.
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(A) Bioavailability. The levels of test substance and relevant
metabolites, as appropriate, should be determined in whole blood, blood
plasma or blood serum at appropriate intervals after initiation of intra-
venous, dermal, and inhalation exposure. The sampling intervals should
be compatible with the exposure route under study. The determinations
need only be done on animals administered the test substance containing
14C-w-hexane or, if the analytical sensitivity is equal or greater, unlabeled
test substance may be used.
(B) Extent of absorption. The total quantities of radioactivity should
be determined for excreta collected daily for 7 days, or until at least 90
percent of theradioactivity has been recovered in the excreta, whichever
occurs first.
(C) Excretion. The quantities of radioactivity eliminated in the urine,
feces, and expired air should be determined separately at time intervals
that provide accurate measurement of clearance and excretory rates. The
collection of carbon dioxide may be discontinued when less than one per-
cent of the dose is found to be exhaled as radioactive carbon dioxide in
24 h.
(D) Tissue distribution. At the termination of each study, the quan-
tities of radioactivity should be determined in blood and in various tissues,
including bone, brain, fat, gastrointestinal tract, gonads, heart, kidney,
liver, lungs, muscle, skin, spleen, thymus, and residual carcass of each
animal.
(E) Change in pharmacokinetics. Results of pharmacokinetics meas-
urements (i.e. biotransformation, extent of absorption, tissue distribution,
and excretion) obtained in rats receiving the single inhalation exposure
to the low dose of the test substance (Group B) should be compared to
the corresponding results obtained in rats receiving repeated inhalation ex-
posures to the low dose of the test substance (Group F).
(ii) Metabolism. At least four animals from each group should be
used for these purposes.
(A) 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 administered.
(B) Changes in biotransformation. Appropriate qualitative and
quantitative assay methods should be used to compare the composition
of radioactive compounds in excreta from rats receiving a single inhalation
exposure (Groups B and C) with that from rats receiving repeated inhala-
tion exposures (Group F).
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(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 also be reported.
(2) Evaluation of results. All data should be evaluated by appropriate
statistical methods.
(3) Reporting results. In addition to the reporting requirements as
specified in 40 CFR part 792, the following information should be re-
ported.
(i) Strain of laboratory animals used.
(ii) Chemical characterization of the test substances, including:
(A) For the radiolabeled test substances, information on the sites and
degree of radiolabeling, including type of label, specific activity, chemical
purity prior to mixing with the unlabeled hexane mixture, and
radiochemical purity.
(B) For the unlabeled test substance, information on lot number and
the percentage of MCP and w-hexane.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy of
all procedures used to obtain the data.
(iv) Percent and rate of absorption of the test substance after inhala-
tion and dermal exposures.
(v) Quantity and percent recovery of radioactivity in feces, urine, ex-
pired air, and blood. For dermal studies, include recovery data for skin
and residual radioactivity in the covering apparatus.
(vi) Tissue distribution reported as quantity of radioactivity in blood,
in various tissues including bone, brain, fat, gastrointestinal tract, gonads,
heart, kidney, liver, lung, muscle, skin, spleen, thymus, and in residual
carcass.
(vii) Biotransformation pathways, to the extent possible, and quan-
tities of the test substances and metabolites in excreta collected after ad-
ministering single high and low doses.
(viii) Biotransformation pathways, to the extent possible, and quan-
tities of test substances and metabolites in excreta collected after admin-
istering repeated low doses.
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(ix) Pharmacokinetics models to the extent they can be developed
from the experimental data.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Susten, A.S. et al. In vivo percutaneous absorption studies of vola-
tile solvents in hairless mice. I. Description of a skin depot. Journal of
Applied Toxicology 6:43-46 (1986).
(2) [Reserved]
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