United States Prevention, Pesticides EPA712-C-96-257
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8500
ToxicokineticTest
'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 870.8500 Toxicokinetic 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 795.235 Toxicokinetic
Test.
(b) Purpose. These studies are designed to determine the
bioavailability of the test substance after dermal or oral treatment; ascertain
whether the metabolites of the test substance are similar after dermal (as-
suming significant penetration) and oral administration; examine the ef-
fects of a multiple dosing regimen on the metabolism of the test substance
after per os administration.
(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.
Absorption toxicokinetics refers to the bioavailability, i.e. the rate and
extent of absorption of the test substance, and metabolism and excretion
rates of the test substance after absorption.
(d) Test procedures—(1) Animal selection—(i) Species. The rat is
the animal species of choice since it has been used extensively for absorp-
tion, metabolism, and toxicological studies.
(ii) Rat strain. Adult male and female Fischer 344 rats should be
used. At 7 to 9 weeks of age, the males should weigh 125 to 175 g and
the females 110 to 150 g. The rats should be purchased from a reputable
dealer and identified with ear tags upon arrival. The animals should be
randomly selected for the testing groups, and no unhealthy animal is to
be used for experimentation.
(iii) Animal care. (A) Animal care and housing should be in accord-
ance with Department of Health, Education and Welfare Publication No.
(NIH)-85-23, 1985. Guidelines for the Care and Use of Laboratory Ani-
mals, or its equivalent.
(B) The animals should be housed in environmentally controlled
rooms with 10 to 15 air changes per hour. The rooms should be maintained
at a temperature of 25 + 2 °C and humidity of 50+ 10 percent with a 12-
h light/dark cycle per day. The rats should be kept in a quarantine facility
for at least 7 days prior to use.
(C) During the acclimatization period, the rats should be housed in
polycarbonate cages on hardwood chip bedding. All animals should be
provided with certified feed and tap water ad libitum.
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(iv) Number of animals. There should be at least four animals of
each sex in each experimental group.
(2) Administration of test substance — (i) Test substance. Test sub-
stance of at least 99 percent purity, commercially available, should be used
as the test substance. Since both nonradioactive and radioactive (uniformly
14C-labeled) test substances are to be used, they should be
chromatographed separately and analyzed together, to ascertain purity and
identity. The use of 14C-labeled test substance, diluted with unlabeled test
substance, is required for all of the studies under this guideline, unless
otherwise specified, as it will greatly increase the reliability and sensitivity
of the quantitative assays and facilitate the identification of metabolites.
(ii) Dosage and treatment. (A) Two doses should be used in studies
under this guideline, a low dose and a high dose. When administered oral-
ly, the high dose level should ideally induce some overt toxicity, such
as weight loss. The low dose level should not induce observable effects
attributable to the test substance. If feasible, the same high and low doses
should be administered orally and dermally.
(B) Oral dosing should be accomplished by gavage after dissolving
the test substance in a suitable vehicle. For dermal treatment, the doses
should be administered in a suitable solvent and applied at a volume ade-
quate to deliver the prescribed doses. The backs of the rats should be
shaved with an electric clipper one day before treatment. The dose should
be applied with a disposable micropipet on a specific area (2 cm2 for rats)
on the shaven skin. The dosed areas should be occluded with an aluminum
foil patch secured in place with adhesive tape.
(iii) Determination of test substance kinetics. Each experimental
group should contain at least four rats of each sex for a total of eight
rats.
(A) Oral studies. (7) Group A should be dosed once orally with the
low dose of the test substance.
(2) Group B should be dosed once orally with the high dose of the
test substance.
For the oral studies, the rats should be placed in individual meta-
bolic cages to facilitate collection of urine and feces at 8, 24, 48, 72, and
96 h following administration. The cages should be cleaned at each time
period to collect any metabolites that might adhere to the metabolic cages.
(B) Dermal studies. (7) Group C should be dosed once dermally with
the low dose of test substance.
(2) Group D should be dosed once dermally with the high dose of
test substance.
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(3)(f) For the dermal studies, the test substance should be applied
for 24 h. Immediately after application, each animal should be placed in
a separate metabolic cage for excreta collection. At the time of removal
of the aluminum foil, the occluded area should be washed with an appro-
priate solvent (see below), to remove any test substance that may be on
the skin surface and the wash solvent assayed for the amount of test sub-
stance recovered. At the termination of the experiments, each animal
should be sacrificed and the exposed skin area removed. The skin (or an
appropriate section) should be solubilized and assayed for the test sub-
stance and its metabolites.
(if) Before initiation of the dermal studies, an initial washing effi-
ciency experiment should be conducted to assess the removal of the ap-
plied test substance by washing the exposed skin area with soap and water
or organic solvents. Four rats, two of each sex, should be lightly anes-
thetized and then test substance applied to a specified area. After applica-
tion (5 to 10 min), the areas should be washed with soap and water (two
rats) or ethanol and water (two rats). The amount recovered should be
determined to assess efficacy of test substance removal by washing of the
skin.
(C) Repeated dosing study Group E. Four rats (two of each sex)
should receive a series of single daily oral doses of nonradioactive test
substance over a period of at least 14 days, followed at 24 h after the
last dose by a single oral dose of 14C-labeled test substance. Each dose
should be at the low dose level.
(3) Observation of animals—(i) Bioavailability—(A) Blood levels.
The levels of 14C should be determined in whole blood, blood plasma,
or blood serum at appropriate intervals from 1 to 96 h after dosing rats
in Groups A through E. Four rats (two of each sex) of each group should
be used for this purpose.
(B) Urinary and fecal excretion. The quantities of 14C excreted in
the urine and feces by rats in Groups A through E should be determined
at 8 h, 24 h, 48 h, 72 h, and 96 h after dosing, and if necessary, daily
thereafter until at least 90 percent of the applied dose has been excreted
or until 7 days after dosing (whichever occurs first). Four animals (two
of each sex) should be used for these analyses.
(ii) Biotransformation after oral and dermal dosing. Appropriate
qualitative and quantitative methods should be used to assay the test sub-
stance and metabolites in the urine and fecal specimens collected from
rat Groups A through D.
(iii) Changes in biotransformation. Appropriate qualitative and
quantitative assay methodology should be used to compare the composition
of 14C-labeled compounds in excreta collected at 14 and 48 h after dosing
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rat Group A with those in the excreta collected at 24 and 48 h after the
14C-labeled test substance dose in the repeated dose study (Group E).
(e) Data and reporting—(1) Treatment of results. Data should be
presented in tabular form.
(2) Evaluation of results. All observed results, quantitative or inci-
dental, should be evaluated by an appropriate statistical method.
(3) Test report. In addition to the reporting requirements specified
in the 40 CFR part 792, subpart J, the following specific information
should be reported:
(i) Species and strains of laboratory animals.
(ii) Information on the degree (i.e. specific activity for a radiolabel)
and sites of labeling of the test substance;
(iii) A full description of the sensitivity and precision of all proce-
dures used to produce the data.
(iv) Percent absorption by oral and dermal routes of rats administered
14C-test substance.
(v) Quantity of isotope, together with percent recovery of adminis-
tered dose in feces, urine, blood, and skin and skin washings (dermal study
only for last two portions).
(vi) Quantity and distribution of 14C-labeled test substance in various
tissues, including bone, brain, fat, gonads, heart, kidney, liver, lung, mus-
cle, spleen, and residual carcass.
(vii) Counting efficacy data should be made available to the Agency
upon request.
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