[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR795.231]
[Page 68-71]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 795_PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart D_Provisional Health Effects Guidelines
Sec. 795.231 Pharmacokinetics of isopropanal.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of the
``test substance'' are similar after oral and inhalation administration.
(2) Determine bioavailability of the test substance after oral and
inhalation administration.
(3) Examine the effects of repeated dosing on the pharmacokinetics
and metabolism of the test substance.
(b) Definitions. (1) ``Bioavailability'' refers to the rate and
relative amount of administered test substance which reaches the
systemic circulation.
(2) ``Metabolism'' means the study of the sum of the processes by
which a
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particular substance is handled in the body, and includes absorption,
tissue distribution, biotransformation, and excretion.
(3) ``Pharmacokinetics'' means the study of the rates of absorption,
tissue distribution, biotransformation, and excretion.
(c) Test procedures--(1) Animal selection--(i) Species. The rat
shall be used because it has been used extensively for metabolic and
toxicological studies.
(ii) Test animals. For pharmacokinetics testing, adult male and
female rats (Fischer 344 or strain used for major toxicity testing), 7
to 9 weeks of age, shall be used. The animals should be purchased from a
reputable dealer and shall be identified upon arrival at the testing
laboratory. The animals shall be selected at random for the testing
groups and any animal showing signs of ill health shall not be used. In
all studies, unless otherwise specified, each test group shall 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
accordance with DHEW Publication No. (NIH)-85-23, 1985, entitled
``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 shall be maintained at
a temperature of 22 2 [deg]C and humidity of 50
20 percent with a 12-hour light/dark cycle per
day. The animals shall be kept in a quarantine facility for at least 7
days prior to use and shall be acclimated to the experimental
environment for a minimum of 48 hours prior to treatment.
(C) During the acclimatization period, the animals should be housed
in suitable cages. All animals shall 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. Ideally, the purity
of both radioactive and nonradioactive test substance should be greater
than 99 percent. The radioactive and nonradioactive substances shall be
chromatographed separately 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, shall be administered
intravenously to four rats of each sex.
(B) Oral. Two doses of test substance shall be used in the oral
portion of the study, a low dose and a high dose. The high dose should
ideally induce some overt toxicity, such as weight loss. The low dose
level should correspond to a no-observed effect level. The oral dosing
shall be accomplished 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 shall be
used in this portion of the study, a low concentration and a high
concentration. The high concentration should ideally induce some overt
toxicity, while the low concentration should correspond to a no observed
level. Inhalation treatment should be conducted using a ``nose-cone'' or
``head only'' apparatus to prevent ingestion of the test substance
through ``grooming''.
(iii) Dosing and sampling schedule. After administration of the test
substance, each rat shall be placed in a separate metabolic unit to
facilitate collection of excreta. For the inhalation studies, excreta
from the rats shall also be collected during the exposure periods. At
the end of each collection period, the metabolic units shall be cleaned
to recover any excreta that might adhere to the cages. All studies,
except the repeated dose study, shall 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 shall be dosed once intravenousely at
the low dose of test substance.
(B) Oral studies. (1) Group B shall be dosed once per os with the
low dose of the test substance.
(2) Group C shall be dosed once per os with the high dose of the
test substance.
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(C) Inhalation studies. A single 6-hour exposure period shall be
used for each group.
(1) Group D shall be exposed to a mixture of the test substance in
air at the low concentration.
(2) Group E shall be exposed to a mixture of test substance in air
at the high concentration.
(D) Repeated dosing study. Group F shall receive a series of single
daily oral low doses of nonradioactive test substance over a period of
at least 7 consecutive days. Twenty four hours after the last
nonradioactive dose, a single oral low dose of radioactive test
substance shall be administered. Following dosing with radioactive
substance, the rats shall be placed in individual metabolic units as
described in paragraph (c)(2)(iii) of this section. The study shall 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 shall 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 shall be measured following
administration. In groups administered the substance by the inhalation
route (i.e., Groups D and E), the concentration of radioactivity in
blood shall be measured at selected time intervals during and following
the exposure period. In the groups administered the substance by
inhalation (i.e., Groups D and E), the concentration of radioactivity in
excreta (including expired air) shall 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 shall be measured at selected time intervals
during the exposure period.
(ii) Metabolism studies. Groups A through F shall be used to
determine the metabolism of the test substance. Excreta (urine, feces,
and expired air) shall be collected for identification and
quantification of test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Bioavailability. The levels of radioactivity shall be determined
in whole blood, blood plasma or blood serum at 15 minutes, 30 minutes,
1, 2, 3, 6, 9, and 18 hours after dosing; and at 30 minutes, 3, 6, 6.5,
7, 8, 9, 12, and 18 hours after initation of inhalation exposure.
(B) Extent of absorption. The total quantities of radioactivity
shall 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.
(C) Excretion. The quantities of radioactivity eliminated in the
urine, feces, and expired air shall be determined separately at
appropriate time intervals. The collection of the intact test substance
or its metabolites, including carbon dioxide, may be discontinued when
less than 1 percent of the administered dose is found to be exhaled as
radioactive carbon dioxide in 24 hours.
(D) Tissue distribution. At the termination of each study, the
quantities 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 shall
be determined.
(E) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., biotransformation, extent of absorption, tissue
distribution, and excretion) obtained in rats receiving the single low
oral dose of test substance (Group B) shall be compared to the
corresponding results obtained in rats receiving repeated oral doses of
test substance (Group F).
(F) Biotransformation. Appropriate qualitative and quantitative
methods shall be used to assay urine, feces, and expired air collected
from rats. Efforts shall be made to identify any metabolite which
comprises 5 percent or more of the dose eliminated.
(G) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall be used to compare the composition
of radioactive substances in excreta from the rats receiving a single
oral dose
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(Groups B and C) with those in the excreta from rats receiving repeated
oral doses (Group F).
(ii) [Reserved]
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetics data shall also be presented in graphical
form. Qualitative observations shall also be reported.
(2) Evaluation of results. All quantitative results shall be
evaluated by an appropriate statistical method.
(3) Reporting results. In addition to the reporting requirements as
specified in the EPA Good Laboratory Practice Standards (40 CFR
792.185), the following specific information shall 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 site(s)
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 inhalation exposure.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood.
(vi) Tissue distribution reported as quantity of radioactivity in
blood and in various tissues, including bone, brain, fat,
gastrointestinal tract, gonads, 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 model(s) developed from the experimental data.
[54 FR 43261, Oct. 23, 1989]