[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: 40CFR795.228]
[Page 65-68]
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. 795.228 Oral/dermal pharmacokinetics.
(a) Purpose. The purposes of these studies are to:
(1) Ascertain whether the pharmacokinetics and metabolism of a
chemical substance or mixture (``test substance'') are similar after
oral and dermal administration.
(2) Determine bioavailability of a test substance after oral and
dermal 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 particular substance is handled in the body and includes absorption,
tissue distribution, biotransformation, and excretion.
(3) Percent absorption means 100 times the ratio between total
excretion of radioactivity following oral or dermal administration and
total excretion following intravenous administration of test substance.
(4) 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 for pharmacokinetics testing because it has been used
extensively for metabolic and toxicological studies. For dermal
bioavailability studies, the rat and the mini-pig shall be used.
(ii) Test animals. For pharmacokinetics testing and dermal studies,
adult male and female Sprague-Dawley rats, 7 to 9 weeks of age, shall be
used. For dermal studies, young adult mini-pigs shall also 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 test 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 4 animals of each sex
for a total of at least 8 animals.
(iii) Animal care. (A) The animals shall be housed in
environmentally controlled rooms with at least 10 air changes per hour.
The rooms shall be maintained at a temperature of 24 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 administration of the test substance.
(B) During the acclimatization period, the animals shall be housed
in suitable cages. All animals shall be
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provided with certified feed and tap water ad libitum. The mini-pig diet
shall 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. Ideally, the
purity, radioactive and nonradioactive, is greater than 99 percent. The
radioactive and nonradioactive test 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 groups of rats and mini-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 shall be used in the oral
study, a low dose and a high dose. The high dose should ideally induce
some overt toxicity, such as weight loss. The low dose should correspond
to a no-observed effect level. The oral dosing shall 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. (1) Dermal treatment. For dermal treatment, two doses,
comparable to the low and high oral doses, shall be dissolved in a
suitable vehicle and applied in volumes adequate to deliver comparable
doses. The backs of the animals should be lightly shaved with an
electric clipper 24 hours before treatment. The test substance shall be
applied to the intact shaven skin (approximately 2 cm\2\ for rats, 5
cm\2\ for mini-pigs). The dosed areas shall be protected with a suitable
porous covering which is secured in place, and the animals shall be
housed separately.
(2) Washing efficacy study. Before initiation of the dermal
absorption studies, an initial washing efficacy experiment shall 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 shall be applied to 4 rats and
4 mini-pigs in accordance with paragraph (c)(2)(ii)(C)(1) of this
section. After application (5 to 10 minutes), the treated areas of 2
rats and 2 mini-pigs shall be washed with soap and water and the treated
areas of the remaining rats and pigs shall be washed with an appropriate
solvent. The amounts of test substance recovered in the washings shall
be determined to assess efficacy of its removal by washing.
(iii) Dosing and sampling schedule--(A) Rat studies. After
administration of the test substance, each rat shall be placed in a
metabolic unit to facilitate collection of excreta. For the dermal
studies, excreta from the rats shall also be collected during the 6 hour
exposure periods. At the end of each collection period, the metabolic
units shall be cleaned to recover any excreta that might adhere to them.
All studies, except the repeated dosing 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.
(1) Intravenous study. Group A shall be dosed once intravenously at
the low dose of test substance.
(2) Oral study. (i) Group B shall be dosed once per os with the low
dose of test substance.
(ii) Group C shall be dosed once per os with the high dose of test
substance.
(3) Dermal studies. Unless precluded by corrosivity, the test
substance shall be applied and kept on the skin for a minimum of 6
hours. At the time of removal of the porous covering, the treated area
shall be washed with an appropriate solvent to remove any test substance
that may be on the skin surface. Both the covering and the washing shall
be assayed to recover residual radioactivity. At the termination of the
studies, each animal shall be sacrificed and the exposed skin area
removed. An appropriate section of the skin shall be solubilized and
assayed for radio-activity to ascertain if the skin acts as a reservoir
for the test substance. Studies on the dermal absorption of corrosive
test substances should be discussed with EPA prior to initiation.
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(i) Group D shall be dosed once dermally with the low dose of test
compound.
(ii) Group E shall be dosed once dermally with the high dose of the
test substance.
(4) 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 days. Twenty-four hours after the last nonradioactive dose, a
single oral low dose of radioactive test substance shall be
administered. Following dosing with the 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 at 7 days
after the last dose, or after at least 90 percent of the radioactivity
has been recovered in the excreta, whichever occurs first.
(B) Mini-Pig studies. For all mini-pig studies, the test groups
shall consist of four young adult animals. After administration of the
test substance, each mini-pig shall 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 shall be terminated at 7 days, or after at
least 90 percent of the radio-activity has been recovered in the
excreta, whichever occurs first.
(1) 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
(c)(2)(iii)(A)(3) of this section:
(i) Group H shall be dosed once dermally with the low dose of test
substance.
(ii) Group I shall be dosed once dermally with the high dose of the
test substance.
(3) Types of studies--(i) Pharmacokinetics studies--(A) Rat studies.
Groups A through F shall be used to determine the kinetics of absorption
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 shall 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 shall be measured at selected time intervals during
and following the exposure period.
(B) Mini-Pig studies. Groups G, H, and I shall be used to determine
the extent of dermal absorption of the test substance. The amount of
radioactivity in excreta shall be determined at selected time intervals.
(ii) Metabolism studies--Rat studies. Groups A through F shall be
used to determine the metabolism of the test substance. Urine, feces,
and expired air shall be collected for identification and quantification
of the test substance and metabolites.
(4) Measurements--(i) Pharmacokinetics. Four animals from each group
shall be used for these purposes.
(A) Rat studies--(1) Bioavailability. The levels of radioactivity
shall be determined in whole blood, blood plasma or blood serum at 15
and 30 minutes and at 1, 2, 8, 24, 48, and 96 hours after initiation of
dosing.
(2) Extent of absorption. The total quantities of radioactivity
shall 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 shall 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 hours.
(4) 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, and residual carcass of each animal shall be
determined.
(5) Changes in pharmacokinetics. Results of pharmacokinetics
measurements (i.e., bioavailability and extent of absorption, tissue
distribution, and excretion) obtained in rats receiving
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the single low oral dose of the test substance (Groups B and C) shall be
compared to the corresponding results obtained in rats receiving
repeated oral doses of the test substance (Group F).
(B) Mini-Pig studies--Extent of absorption. The total quantities of
radioactivity shall 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 shall be used for
these purposes.
(A) Rat studies--(1) 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 administered dose
and the major radioactive components of blood.
(2) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology shall 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).
(d) Data and reporting. The final test report shall include the
following:
(1) Presentation of results. Numerical data shall be summarized in
tabular form. Pharmacokinetic 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 40 CFR part 792, 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 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 purity.
(C) Results of chromatography.
(iii) A full description of the sensitivity, precision, and accuracy
of all procedures used to generate the data.
(iv) Percent of absorption of test substance after oral and dermal
exposures to rats and dermal exposure to mini-pigs.
(v) Quantity and percent recovery of radioactivity in feces, urine,
expired air, and blood. In dermal studies on rats and mini-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, gonads, 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 model(s) developed from the experimental data.
[54 FR 33411, Aug. 14, 1989; 54 FR 49844, Dec. 1, 1989; 55 FR 25392,
June 21, 1990]