United States Prevention, Pesticides EPA712-C-98-244
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.7485
Metabolism and
Pharmacokinetics
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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.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World Wide Web site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test
Guidelines."
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OPPTS 870.7485 Metabolism and pharmacokinetics
(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 materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798.7485 Metabolism and
pharmacokinetics; OPP 85-1 Metabolism Study (Pesticide Assessment
Guidelines, Subdivision F - Hazard Evaluation: Human and Domestic Ani-
mals, EPA report 540/09-82-025, October 1982); and OECD Guideline
417 Toxicokinetics.
(b) Purpose. (1) Testing of the disposition of a test substance is de-
signed to obtain adequate information on its absorption, distribution, bio-
transformation, and excretion and to aid in understanding the mechanism
of toxicity. Basic pharmacokinetic parameters determined from these stud-
ies will also provide information on the potential for accumulation of the
test substance in tissues and/or organs and the potential for induction of
biotransformation as a result of exposure to the test substance. These data
can be used to assess the adequacy and relevance of the extrapolation of
animal toxicity data (particularly chronic toxicity and/or carcinogenicity
data) to human risk assessment.
(2) Metabolism data can also be used to assist in determining whether
animal toxicity studies have adequately addressed any toxicity concerns
arising from exposure to plant metabolites, and in the setting of tolerances,
if any, for those metabolites in raw agricultural commodities.
(c) Definitions. The following definitions apply to this guideline:
Metabolism (biotransformation) is the sum of the processes by which
a foreign chemical is subjected to chemical change by living organisms.
LOEL is the lowest observable effect level.
NOEL is the no observable effect level.
Pharmacokinetics is the quantitation and determination of the time
course and dose dependency of the absorption, distribution, biotrans-
formation, and excretion of chemicals.
(d) Good laboratory practice standards. The pharmacokinetics and
metabolism tests outlined in this guideline are to conform to the laboratory
practices stipulated in 40 CFR parts 160 and 792 (Good Laboratory Prac-
tice Standards).
(e) Test Procedures. Test procedures presented below utilize a tier
system to minimize the use of resources and to allow flexibility in the
conduct of metabolism studies. The proposed tier system will consist of
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a basic data set (Tier 1) and additional studies (Tier 2) which may be
requested based upon the existing toxicology data base and/or the results
of Tier 1 testing which are found to impact upon the risk assessment proc-
ess. For Tier 1 testing, the oral route will typically be required; however,
if the use pattern results in other types of exposure, other routes (dermal
and/or inhalation) may be required for initial testing of the disposition of
a chemical substance. The registrant should justify the route of exposure
to the Agency. Complete descriptions of the test procedures for these other
routes of exposure can be found under paragraph (i) of this guideline. Ex-
cept in unusual circumstances, the tiered approach to metabolism testing
should apply to all listed routes of exposure.
(1) Pilot studies. The use of pilot studies is recommended and en-
couraged for the selection of experimental conditions for the
pharmacokinetics and metabolism studies (mass balance, analytical proce-
dures, dose-finding, excretion of CCh, etc.).
(2) Animal selection—(i) Species. The rat shall normally be used
for testing because it has been used extensively for metabolic and toxi-
cological studies. The use of other or additional species may be required
if critical toxicology studies demonstrate evidence of significant toxicity
in these species or if metabolism is shown to be more relevant to humans
in the test species.
(ii) Strain. Adult animals of the strain used or proposed to be used
for the determination of adverse health effects associated with the test sub-
stance.
(3) Material to be tested—(i) Test substance. (A) A radiolabeled
test substance using 14C should be used for all material balance and
metabolite identification aspects of the study. Other radioactive and stable
isotopes may be used, particularly if the element is responsible for or is
a part of the toxic portion of the compound. If it can be demonstrated
that the material balance and metabolite identification requirements can
be met using unlabeled test substance, then radiolabeled compound need
not be used. If possible, the radiolabel should be located in a core portion
of the molecule which is metabolically stable (it is not exchangeable, is
not removed metabolically as CCh, and does not become part of the one-
carbon pool of the organism). Labeling of multiple sites of the molecule
may be necessary to follow the metabolic fate of the compound.
(B) The label should follow the test compound and/or its major
metabolites until excreted. The radiopurity of the radioactive test substance
shall be the highest attainable for a particular test substance (ideally it
should be greater than 95 percent) and reasonable effort should be made
to identify impurities present at or above 2 percent. The purity, along with
the identity of major impurities which have been identified, shall be re-
ported. For other segments of the study, nonradioactive test substance may
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be used if it can be demonstrated that the analytical specificity and sen-
sitivity of the method used with nonradioactive test substance is equal to
or greater than that which could be obtained with the radiolabeled test
substance. The radioactive and nonradioactive test substances shall be ana-
lyzed using an appropriate method to establish purity and identity. Addi-
tional guidance will be provided in chemical specific test rules to assist
in the definition and specifications of test substances composed of mixtures
and methods for determination of purity.
(ii) Administration of test substance. Test substance should be dis-
solved or suspended homogeneously in a vehicle usually employed for
acute administration. A rationale for the choice of vehicle should be pro-
vided. The customary method of administration will be by oral gavage;
however, administration by gelatin capsule or as a dietary mixture may
be advantageous in specific situations. Verification of the actual dose ad-
ministered to each animal should be provided.
(4) Tier testing, (i) The multiplicity of metabolic parameters that im-
pact the outcome of toxicological evaluations preclude the use of a univer-
sal study design for routine toxicological evaluation of a test substance.
The usefulness of a particular study design depends upon the biological
activity of a compound and circumstances of exposure. For these reasons,
a tiered system is proposed for evaluation of the metabolism/kinetic prop-
erties of a test substance.
(ii) The first tier data set is a definitive study by the appropriate route
of exposure conducted in male rats to determine the routes and rate of
excretion and to identify excreted metabolites. First tier data will also pro-
vide basic information for additional testing (Tier 2) if such testing is con-
sidered necessary. In the majority of cases, Tier 1 data are expected to
satisfy regulatory requirements for biotransformation and pharmacokinetic
data on test chemicals.
(iii) Second tier testing describes a variety of metabolism/kinetic ex-
periments which address specific questions based on the existing toxi-
cology data base and/or those results of Tier 1 testing impacting signifi-
cantly on the risk assessment process. For conduct of these studies, indi-
vidualized protocols may be necessary. Protocols for these studies, if re-
quired, can be developed as a cooperative effort between Agency and in-
dustry scientists.
(f) Tier 1 data requirements (minimum data set). At this initial
level of testing, biotransformation and pharmacokinetic data from a single
low dose group will be required. This study will determine the rate and
routes of excretion and the type of metabolites generated.
(1) Number and sex of animals. A minimum of four male young
adult animals will be required for Tier 1 testing. The use of both sexes
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may be required in cases where there is evidence to support significant
sex-related differences in toxicity.
(2) Dose selection, (i) A single dose is required for each route of
exposure. The dose should be nontoxic, but high enough to allow for
metabolite identification in excreta. If no other toxicity data are available
for selection of the low dose, a dose identified as a fraction of the LD50
(as determined from acute toxicity studies) may be used. The magnitude
of the dose used in Tier 1 studies should be justified in the final report.
(ii) For test substances of low toxicity a maximum dose of
1,000 mg/kg should be used; chemical-specific considerations may neces-
sitate a higher maximum dose and will be addressed in specific test rules.
(3) Measurements—(i) Excretion. (A) Data obtained from this sec-
tion (percent recovery of administered dose from urine, feces, and expired
air) will be used to determine the rate and extent of excretion of test chem-
ical, to assist in establishing mass balance, and will be used in conjunction
with pharmacokinetic parameters to determine the extent of absorption.
The quantities of radioactivity eliminated in the urine, feces, and expired
air shall be determined separately at appropriate time intervals.
(B) If a pilot study has shown that no significant amount of radio-
activity is excreted in expired air, then expired air need not be collected
in the definitive study.
(C) Each animal is to be placed in a separate metabolic unit for col-
lection of excreta (urine, feces and expired air). At the end of each collec-
tion period, the metabolic units are to be rinsed with appropriate solvent
to ensure maximum recovery of radiolabel. Excreta collection shall be ter-
minated at 7 days, or after at least 90 percent of the administered dose
has been recovered, whichever occurs first. The total quantities of radio-
activity in urine are to be determined at 6, 12, and 24 h on day 1 of
collection, and daily thereafter until study termination, unless pilot studies
suggest alternate or additional time points for collection. The total quan-
tities of radioactivity in feces should be determined on a daily basis begin-
ning at 24 h post-dose, and daily thereafter until study termination. The
collection of CC>2 and other volatile materials may be discontinued when
less than 1 percent of the administered dose is found in the exhaled air
during a 24-h collection period.
(ii) Tissue distribution. At the termination of the Tier 1 study, the
following tissues should be collected and stored frozen: Liver, fat, gastro-
intestinal tract, kidney, spleen, whole blood, and residual carcass. If it is
determined that a significant amount of the administered dose is unac-
counted for in the excreta, then data on the percent of the total (free and
bound) radioactive dose in these tissues as well as residual carcass will
be requested. Additional tissues shall be included if there is evidence of
target organ toxicity from subchronic or chronic toxicity studies. For other
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routes of exposure, specific tissues may also be required, such as lungs
in inhalation studies and skin in dermal studies. Certain techniques cur-
rently at various stages of development, e.g. quantitative whole-body
autoradiography, may prove useful in determining if a test substance con-
centrates in certain organs or in determining a specific pattern of distribu-
tion within a given tissue. The use of such techniques is encouraged, but
not required, and may be employed to limit the number of tissues collected
to those shown to contain a measurable amount of radioactivity.
(iii) Metabolism. Excreta shall be collected for identification and
quantitation of unchanged test substance and metabolites as described
under paragraph (f)(3)(i) of this guideline. Pooling of excreta to facilitate
metabolite identification within a given dose group is acceptable. Profiling
of metabolites from each time period is recommended. However, if lack
of sample and/or radioactivity precludes this, pooling of urine as well as
pooling of feces across several time points is acceptable. Appropriate qual-
itative and quantitative methods shall be used to assay urine, feces, and
expired air from treated animals. Reasonable efforts should be made to
identify all metabolites present at 5% or greater of the administered dose
and to provide a metabolic scheme for the test chemical. Compounds
which have been characterized in excreta as comprising 5 percent or great-
er of the administered dose should be identified. If identification at this
level is not possible, a justification/explanation should be provided in the
final report. Identification of metabolites representing less than 5 percent
of the administered dose might be requested if such data are needed for
risk assessment of the test chemical. Structural confirmation should be pro-
vided whenever possible. Validation of the methods used in metabolite
identification should be included.
(g) Tier 2 data requirements. Studies at the Tier 2 level are designed
to answer questions about the disposition of test chemicals based on the
existing toxicology data base and/or results of Tier 1 testing which may
have a significant impact on the risk assessment for the test chemical.
Such studies may address questions regarding absorption, persistence, or
distribution of the test chemical, or a definitive alteration in the metabolic
profile occurring with dose which may be of toxicological concern. At
the Tier 2 level, only those studies which address a specific concern are
required, and will be conducted according to mutual agreement between
the registrant and the Agency. Flexibility will be allowed in the design
of specific experiments as warranted by technological advances in this
field.
(1) Absorption, (i) If the extent of absorption cannot be established
from Tier 1 studies, or where greater than 20 percent of the administered
dose is present in feces, a study to determine the extent of absorption
will be required. This can be accomplished either through intravenous ad-
ministration of test material and measurement of radioactivity in excreta
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or after oral administration of test material and measurement of radioactiv-
ity in bile.
(ii) For the intravenous study, a single dose (not to exceed the oral
dose used in Tier 1) of test chemical using an appropriate vehicle should
be administered in a suitable volume (e.g. 1 mL/kg) at a suitable site to
at least three male rats (both sexes might be used if warranted). The dis-
position of the test chemical should be monitored for oral dosing as out-
lined in paragraph (f)(3)(i) of this guideline. Metabolite identification will
not be required for this study.
(iii) If a biliary excretion study is chosen the oral route of administra-
tion may be requested. In this study, the bile ducts of at least three male
rats (or of both sexes, if warranted) should be appropriately cannulated
and a single dose of the test chemical should be administered to these
rats. Following administration of the test chemical, excretion of radioactiv-
ity in bile should be monitored as long as necessary to determine if a
significant percentage of the administered dose is excreted via this route.
(2) Tissue distribution time course, (i) A time course of tissue dis-
tribution in selected tissues may be required to aid in the determination
of a possible mode of toxic action. This concern may arise from evidence
of extended half-life or possible accumulation of radioactivity in specific
tissues. The selection of tissues for this type of study will be based upon
available evidence of target organ toxicity and/or carcinogenicity, and the
number of time points required will be based upon pharmacokinetic infor-
mation obtained from Tier 1 data. Flexibility will be allowed in the selec-
tion of time points to be studied.
(ii) For this type of study, three rats per time point will be adminis-
tered an appropriate oral dose of test chemical, and the time course of
distribution monitored in selected tissues. Only one sex may be required,
unless target organ toxicity is observed in sex-specific organs. Assessment
of tissue distribution will be made using appropriate techniques for assess-
ment of total amount distributed to tissue and for assessment of metabolite
distribution.
(3) Plasma kinetics. The purpose of this experiment is to obtain esti-
mates of basic pharmacokinetic parameters (half-life, volume of distribu-
tion, absorption rate constant, area under the curve) for the test substance.
Kinetic data may be required if the data can be used to resolve issues
about bioavailability and to clarify whether clearance is saturated in a
dose-dependent fashion. For this experiment a minimum of three rats per
group is required. At least two doses will be required, usually the NOEL
and LOEL from the critical toxicology study. Following administration
of test substance, samples should be obtained from each animal at suitable
time points appropriate sampling methodology. Total radioactivity present
(or total amount of chemical, for nonradioactive materials) should be ana-
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lyzed in whole blood and plasma using appropriate methods, and the
blood/plasma ratio should be calculated.
(4) Induction, (i) Studies addressing possible induction of biotrans-
formation may be requested under one or more of the following conditions:
(A) Available evidence indicates a relationship between induced me-
tabolism and enhanced toxicity.
(B) The available toxicity data indicate a nonlinear relationship be-
tween dose and metabolism.
(C) The results of Tier 1 metabolite identification studies show identi-
fication of a potentially toxic metabolite.
(D) Induction can plausibly be invoked as a factor in such effects
where status may depend on the level of inducible enzymes present. Sev-
eral in vivo and in vitro methods are available for assessment of enzyme
induction, and the experiments which best address the issue at hand can
be determined between Agency and industry scientists. If induction is dem-
onstrated, the relationship of this phenomenon to toxicity observed from
subchronic and/or chronic toxicity studies will need to be addressed.
(iii) If toxicologically significant alterations in the metabolic profile
of the test chemical are observed through either in vitro or in vivo experi-
ments, characterization of the enzyme(s) involved (for example, Phase I
enzymes such as isozymes of the Cytochrome P450-dependent mono-
oxygenase system, Phase II enzymes such as isozymes of sulfotransferase
or uridine diphosphate glucuronosyl transferase, or any other relevant en-
zymes) may be requested. This information will help establish the rel-
evance of the involved enzyme(s) to human risk, as it is known that certain
isozymes are present in animal species which are not present in humans,
and vice versa.
(5) Physiologically-based modeling. Traditional methods of model-
ing have been used to determine kinetic parameters associated with drug
and xenobiotic disposition, but have assumed a purely mathematical con-
struct of mammalian organisms in their operation. On the other hand, more
recent models which take into account the physiological processes of the
animal have been used with success in defining biological determinants
of chemical disposition as well as the relationship between tissue dose
and tissue response. These so-called physiologically-based models, also
allow for cross-species extrapolation which is often necessary in the risk-
assessment process. The use of physiologically-based modeling as an ex-
perimental tool for addressing specific issues related to biotransformation
and pharmacokinetics of a test substance is encouraged. Information as
derived from physiologically-based modeling experiments may aid in the
comparison of biotransformation and pharmacokinetics of a test substance
between animal species and humans, and in the assessment of risk under
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specific exposure conditions. At the discretion of the Agency, or by mutual
agreement, results of physiologically based pharmacokinetic (PBPK) stud-
ies with parent compound may be submitted in lieu of other studies, if
it is determined that such data would provide adequate information to sat-
isfy this guideline.
(h) Reporting of study results. In addition to the reporting require-
ments specified in 40 CFR part 792, subpart J, the completed study (Tier
1 or Tier 2) should be presented in the following format:
(1) Title/Cover Page. Title page and additional requirements (require-
ments for data submission, good laboratory practice, statements of data
confidentiality claims and quality assurance) if relevant to the study report,
should precede the content of the study formatted below. These require-
ments are to be found in 40 CFR parts 158 and 160 or parts 790, 792,
and 799.
(2) Table of Contents. A concise listing is to precede the body of
the report, containing all essential elements of the study and the page and
table number where the element is located in the final report of the study.
Essential elements of the Table of Contents should include a summary,
an introduction, the materials and methods section, results, discussion/ con-
clusions, references, tables, figures, appendices, and key subsections as
deemed appropriate. The Table of Contents should include the page num-
ber of each of these elements.
(3) Body of the report. The body of the report shall include informa-
tion required under this guideline, organized into sections and paragraphs
as follows:
(i) Summary. This section of the study report is to contain a summary
and analysis of the test results and a statement of the conclusions drawn
from the analysis. This section should highlight the nature and magnitude
of metabolites, tissue residue, rate of clearance, bioaccumulation potential,
sex differences, etc. The summary should be presented in sufficient detail
to permit independent evaluation of the findings.
(ii) Introduction. This section of the report should include the objec-
tives of the study, guideline references, regulatory history, if any, and a
rationale.
(iii) Materials and methods. This section of the report is to include
detailed descriptions of all elements including:
(A) Test substance. (7) This section should include identification of
the test substance—chemical name, molecular structure, qualitative and
quantitative determination of its chemical composition, and type and quan-
tities of any impurities whenever possible.
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(2) This section should also include information on physical prop-
erties including physical state, color, gross solubility and/or partition coef-
ficient, and stability.
The type or description of any vehicle, diluents, suspending
agents, and emulsifiers or other materials used in administering the test
substance should be stated.
(4) If the test substance is radiolabeled, information on the following
should be included in this subsection: The type of radionuclide, position
of label, specific activity, and radiopurity.
(B) Test animals. This section should include information on the test
animals, including: Species, strain, age at study initiation, sex, body
weight, health status, and animal husbandry.
(C) Methods. This subsection should include details of the study de-
sign and methodology used. It should include a description of:
(7) How the dosing solution was prepared and the type of solvent,
if any, used.
(2) Number of treatment groups and number of animals per group.
(3) Dosage levels and volume.
(4) Route of administration.
(5) Frequency of dosing.
(6) Fasting period (if used).
(7) Total radioactivity per animal.
($) Animal handling.
(9) Sample collection.
(10) Sample handling.
(11} Analytical methods used for separation.
(72) Quantitation and identification of metabolites.
(13) Other experimental measurements and procedures employed (in-
cluding validation of test methods for metabolite analysis).
(D) Statistical analysis. If statistical analysis is used to analyze the
study findings, then sufficient information on the method of analysis and
the computer program employed should be included so that an independent
reviewer/statistician can reevaluate and reconstruct the analysis. Presen-
tation of models should include a full description of the model to allow
independent reconstruction and validation of the model.
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(iv) Results. All data should be summarized and tabulated with appro-
priate statistical evaluation and placed in the text of this section. Radio-
activity counting data should be summarized and presented as appropriate
for the study, typically as disintegrations per minute and microgram or
milligram equivalents, although other units may be used. Graphic illustra-
tions of the findings, reproduction of representative chromatographic and
spectrometric data, and proposed metabolic pathways and molecular struc-
ture of metabolites should be included in this section. In addition the fol-
lowing information is to be included in this section if applicable:
(A) Justification for modification of exposure conditions, if applica-
ble.
(B) Justification for selection of dose levels for pharmacokinetic and
metabolism studies.
(C) Description of pilot studies used in the experimental design of
the pharmacokinetic and metabolism studies, if applicable.
(D) Quantity and percent recovery of radioactivity in urine, feces,
and expired air, as appropriate. For dermal studies, include recovery data
for treated skin, skin washes, and residual radioactivity in the covering
apparatus and metabolic unit as well as results of the dermal washing
study.
(E) Tissue distribution reported as percent of administered dose and
microgram equivalents per gram of tissue.
(F) Material balance developed from each study involving the assay
of body tissues and excreta.
(G) Plasma levels and pharmacokinetic parameters after administra-
tion by the relevant routes of exposure.
(H) Rate and extent of absorption of the test substance after adminis-
tration by the relevant routes of exposure.
(I) Quantities of the test substance and metabolites (reported as per-
cent of the administered dose) collected in excreta.
(J) Individual animal data.
(v) Discussion and conclusions. (A) In this section the author(s)
should:
(7) Provide a plausible explanation of the metabolic pathway for the
test chemical.
(2) Emphasize species and sex differences whenever possible.
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(3) Discuss the nature and magnitude of metabolites, rates of clear-
ance, bioaccumulation potential, and level of tissue residues as appropriate.
(B) The author(s) should be able to derive a concise conclusion that
can be supported by the findings of the study.
(vi) Optional sections. The authors may include additional sections
such as appendices, bibliography, tables, etc.
(i) Alternate routes of exposure for Tier 1 testing—(1) Dermal—
(i) Dermal treatment. One (or more if needed) dose levels of the test
substance shall be used in the dermal portion of the study. The low dose
level should be selected in accordance with paragraph (f)(2) of this guide-
line. The dermal doses shall be dissolved, if necessary, in a suitable vehicle
and applied in a volume adequate to deliver the doses. Shortly before test-
ing, fur is to be clipped from the dorsal area of the trunk of the test ani-
mals. Shaving may be employed, but it should be carried out approxi-
mately 24 h before the test. When clipping or shaving the fur, care should
be taken to avoid abrading the skin, which could alter its permeability.
Approximately 10 percent of the body surface should be cleared for appli-
cation of the test substance. With highly toxic substances, the surface area
covered may be less than approximately 10 percent, but as much of the
area as possible is to be covered with a thin and uniform film. The same
nominal treatment surface area shall be used for all dermal test groups.
The dosed areas are to be protected with a suitable covering which is
secured in place. The animals shall be housed separately.
(ii) Dermal washing study. (A) A washing experiment is to be con-
ducted to assess the removal of the applied dose of the test substance
by washing the treated skin area with a mild soap and water. A single
dose shall be applied to two animals in accordance with paragraph (f)(2)
of this guideline. After application (2 to 5 min) the treated areas of the
animals shall be washed with a mild soap and water. The amounts of test
substance recovered in the washes shall be determined to assess the effec-
tiveness of removal by washing.
(B) Unless precluded by corrosiveness, the test substance shall be ap-
plied and kept on the skin for a minimum of 6 h. At the time of removal
of the covering, the treated area shall be washed following the procedure
as outlined in the dermal washing study. Both the covering and the washes
shall be analyzed for residual test substance. At the termination of the
studies, each animal shall be sacrificed and the treated skin removed. An
appropriate section of treated skin shall be analyzed to determine residual
radioactivity.
(2) Inhalation. A single (or more if needed) concentration of test
substance shall be used in this portion of the study. The concentration
should be selected in accordance with paragraph (f)(2) of this guideline.
Inhalation treatments are to be conducted using a "nose-cone" or "head-
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only" apparatus to prevent absorption by alternate routes of exposure. If
other inhalation exposure conditions are proposed for use in a chemical-
specific test rule, justification for the modification must be documented.
A single exposure over a defined period shall be used for each group—
a typical exposure is 4-6 h.
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