United States Prevention, Pesticides EPA712-C-96-350
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.7600
Dermal Penetration
'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 870.7600 Dermal Penetration.
(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. This guideline was developed in the Office of Pes-
ticide Programs of and published as OPP 85-3 Dermal Absorption Studies
of Pesticides (Pesticide Assessment Guidelines, Subdivision F—Hazard
Evaluation; Human and Domestic Animals) EPA report 540/09-82-025,
1982. Dermal absorption studies are not routinely required.
(b) Discussion. (1) Dermal absorption studies are complex kinetic
studies which, in and of themselves, provide no information on the biologi-
cal activity (toxicity) of a compound. Dermal absorption studies may be
required on an individual basis for compounds having a serious toxic effect
identified by oral or inhalation studies, for which a significant route of
human exposure is dermal and for which the assumption of 100 percent
dermal absorption does not produce an adequate margin of exposure
(MOE). That is, a risk assessment must be performed to determine the
need for a dermal absorption study. Dermal absorption studies cannot sub-
stitute for general dermal toxicity studies of up to 90 days of dosing
(OPPTS 870.3200 and OPPTS 870.3250), which must be performed by
the dermal route to assess direct toxicity to the skin (and systemic toxicity
as well).
(2) It is recommended that studies showing significant toxicological
effects, first identified by the oral or inhalation route, be repeated by the
dermal route where practical, rather than performing a dermal absorption
study. Examples are studies of organ, system, or physiological process spe-
cific short term toxicity such as developmental toxicity, immunotoxicity,
or neurotoxicity studies. Low-effect and no-effect doses from such studies
can be used directly in the calculation of and MOE without the necessity
of kinetic evaluations. This approach is preferred since the dermal route
may produce differences in distribution, metabolism, storage, and excretion
from the oral or inhalation routes which can produce qualitative as well
as quantitative differences in the toxicity of a compound. Such differences
are not easily identified by kinetic or metabolism studies and can have
significant effects on the systemic toxic response.
(3) Information on dermal exposure is necessary in order to determine
doses and durations of exposure to be evaluated in the dermal absorption
study. It is expected that this information will have been gathered in order
to perform the risk assessment and to make the basic decision as to wheth-
er the study is required.
(4) An oral kinetic study in the rat is strongly recommended in order
to allow full utilization of the dermal kinetic study in risk assessment.
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It is recommended that the oral kinetic study be performed before the der-
mal absorption study is undertaken. The study must determine the portion
(percent) of the oral dose that is absorbed and the rate of absorption, the
rate of tissue distribution, the routes and rate of excretion and be sufficient
to allow half-life determinations for each of the rates.
(c) Purpose. Data from dermal absorption studies allow the Agency
to make risk determinations in cases where the toxic effect has been deter-
mined by the oral or inhalation route in the experimental animal and the
exposure to humans is by the dermal route. Such determinations may vary
in degree of complexity ranging from the 100 percent assumption used
to justify performing the dermal absorption study to a complete kinetic
analysis. A complete kinetic analysis essentially enables an investigator
to convert oral or inhalation low-effect and no-effect doses into dermal
low-effect and no-effect doses, thus allowing the calculation of an MOE
or risk for systemic toxic effects which have not been or cannot be tested
practically by the dermal route. The analysis can be performed in several
ways. The first involves calculating the maximum systemic doses produced
by the oral (or inhalation) no-effect and low-effect doses and calculating
dermal doses that will produce the same maximum systemic doses. The
calculated dermal no-effect and low-effect values are then compared with
the dermal exposures from the risk assessment. In a second method the
dermal exposure is converted to a maximum systemic dose which is com-
pared with the maximum systemic doses equivalent to the oral no-effect
and low-effect doses.
(d) Material to be tested—(1) Compound. The compound should
be of known chemical purity and radiolabeled, usually with 14C, in a posi-
tion which is part of the "core" of the compound. The label should follow
the compound and its major metabolites until excreted. To the extent prac-
tical, the label should not be exchangable nor should it be metabolically
removed to C02 or become part of the one-carbon pool of the organism.
Other radioactive isotopes such as 35S, 36C1, and 113Sn or stable isotopes
such as 15N and 18O may be used, particularly if the element is responsible
for, or is a part of, the toxic portion of the compound. Labeled compound
may not be required if a sufficiently selective and sensitive physical/chemi-
cal test for identifying the compound is used. The specificity and sensitiv-
ity of the test must be demonstrated in the biological systems (organs,
tissues, and body fluids) being analyzed and a report of this validation
must be included in the report of the dermal absorption study. Use of
a physical/chemical test may require the use of control (untreated) animals
if background interference with the procedure is observed.
(2) Vehicle/solvent. The vehicle system used should duplicate that
under which field exposure occurs. The basic vehicle is usually the mate-
rial used in the commercial formulation (formulation blank). Dilutions are
made with the field vehicle, usually water, to produce a solution or suspen-
sion. In cases where exposure is to the chemical and not a formulation
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or dilution thereof, a neutral suspending agent which does not interact with
the test chemical or affect the permeability of the skin, such as
carboxymethylcellulose, may be used. However, organic solvents or spe-
cial solubilizing/suspending agents must not be used. Formulations applied
dry (dusts, granulars, etc.) should be moistened with water to assist in
the application a quantitative dose. This moisture mimics to some measure
the presence of perspiration in field use.
(3) Dose preparation. Dose solutions/suspensions should be prepared
so that sufficient quantities of radiolabel are present in each dose to allow
sufficient sensitivity for that dose. The sensitivity required for a dose de-
pends upon how small a quantity of the test compound is considered nec-
essary to produce a toxic effect. If it is not possible to detect a significantly
small fraction of that quantity, it will not be possible to determine if the
quantity absorbed allows an acceptable MOE or produces a toxicologically
acceptable risk. The dosing solutions/suspensions may be obtained by any
practical and reliable method that the laboratory can devise. Predictability
of dose preparation is most important—it is critical to know the actual
concentrations obtained and that the dosing material is homogeneous so
that the actual dose administered is known. It is not critical that the nomi-
nal dose be precisely administered.
(e) Test procedure—(1) Variation of procedure. The basic study
described is designed to cover the entire range of doses and durations of
exposure expected for a pesticide designed for a wide variety of uses. It
is frequently possible to cover the use pattern at risk for a particular pes-
ticide with a lesser number of doses and durations of exposure. In the
case of pesticides having a limited pattern of use, Registrants may, after
consultation with the Agency, perform only those doses and durations of
exposure that are applicable to the use pattern which is being considered
for risk assessment.
(2) Test animal—(i) Species and strain. The laboratory rat is the
required species because this guideline has been designed and validated
for the rat. Other animal species have been considered and rejected. It
is recommended that the rats be of the same strain as those used for the
metabolism and toxicology studies on the test compound. The rat is not
intended as a model of absorption through the human skin but rather as
a test system for dermal absorption. It is possible to use the absorption
rates determined as a modest overestimate of human dermal absorption
or to perform a kinetic evaluation as described in paragraph (c) of this
guideline for risk assessment purposes.
(ii) Age. Young adult animals should be used.
(iii) Sex. The male rat should be used. The choice of a single sex
allows consistency and comparison from study to study.
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(iv) Numbers. A total of 24 animals is used at each dose level. This
is based on four animals per dose per exposure duration, the minimal ex-
perimental unit. Studies using fewer exposure durations will use fewer ani-
mals per dose but the same number (four) per duration of exposure.
(3) Dose levels and dose selection, (i) Four doses are recommended;
at least three doses should be used. Experience has shown that absorption
is not dose-linear—as the dose per unit area increases the flux increases
to a lesser degree. Some compounds have shown saturation of the absorp-
tion process (no increase of flux with dose). Thus, the quantity and rate
of absorption determined at one dose is generally not valid at a dose which
is greater or less by more than approximately one-half of a log unit. Com-
pounds that are caustic or are dermal irritants should be tested only at
doses which do not show such effects. Direct dermal toxicity is considered
an unacceptable risk and compounds which show such effects are regulated
using the low-effect and no-effect doses for direct dermal toxicity.
(ii) Doses should be at log intervals (i.e. 1.0, 0.1, 0.01 and
0.001 mg/cm2) and span the range of doses expected in field exposure.
The numbers are examples—the interval is the important factor. The maxi-
mum practical dose is on the order of 1 mg/cm2—larger doses tend to
fall off the skin or exceed saturation of the absorption process. When only
three doses are qiven the highest dose should be on the order of
0.1 mg/cm2. When different forms of the compound are tested (salts,
esters, etc.), comparative doses should be equimolar to the doses of the
parent compound.
(iii) Doses must be determined on the basis of quantity per unit area
of exposed skin (expressed as milligrams per square centimeter, not on
the basis of quantity per unit of body weight (milligrams per kilogram).
In this study the skin is being dosed to determine its permeability to the
test compound which is dependent on the dose to the skin. The test animal
per se is not being dosed.
(iv) The maximum dose volume should not exceed 10 (iL/cm2. Larger
volumes of liquid have been found to flow on the skin and produce uneven
dose distribution on the dosed area.
(4) Duration of exposure. In the full study four animals per dose
are exposed for durations of 0.5, 1, 2, 4, 10, and 24 h. For an abbreviated
study, designed for a single exposure scenario, the recommended minimal
durations of exposure are 1, 10, and 24 h. The evaluation with time is
recommended since experience has shown that skin deposition (wash-re-
sistant) and penetration are rarely linear with time, the greatest variation
occurring in the first 1 or 2 h of exposure.
(5) Administration of the test substance—(i) Animal preparation.
The back and shoulders of the rats are clipped free of hair and the area
wiped with acetone 24 h prior to dosing. A soap and water wash may
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be used but care must be taken to rinse the area throughly to remove resid-
ual soap. A soap and water wash is generally harder on the skin than
an acetone wipe. Clipping and wipe produce a standardized skin condition
at the time of dosing. Use of a shorter interval between skin wipe and
dosing has been shown to increase dermal penetration. Animals snowing
damage to the skin at the time of dosing should not be used.
(ii) Dose application. The measured dose of the compound is applied
to a measured area of the rat's skin of no less than 10 cm2. Because most
dose forms are suspensions, this minimal area is necessary for even spread-
ing. The material is spread evenly until a film is formed over the applica-
tion site. The spreader should be checked for retention of material and
the actual dose applied should be determined by subtraction of retained
material from the total dose. Particular attention must be paid to determin-
ing the actual dose applied.
(iii) Site protection. The application area must be covered to prevent
loss of compound through falling off, being rubbed off, or being licked.
The cover must allow air circulation over the application site to allow
normal evaporation of surface water from the skin. A combination cover
(protective device) consisting of a spacer (a rubber, plastic or glass rectan-
gle, square, or ring glued to the skin) to outline the application site and
a filter paper or gauze cover glued to it is recommended. The spacer should
be impervious to the test solution. The paper or gauze should not be in
direct contact with the test material or the skin. See paragraph (g)(5) of
this guideline for information on volatile chemicals.
(6) Animal processing. The treated animals are placed individually
in metabolism cages. Expired air should be collected if a metabolism study
shows that label is expired. Total urine and feces are collected separately
(a single collection for the entire duration of exposure). At the exposure
intervals (0.5, 1, 2, 4, 10 and 24 h for the standard study) four animals
per dose are anesthetized, exposed skin is washed with a mild soap/deter-
gent solution followed by several water rinses, to mimic human washing,
and the protective device is removed. The skin at the exposure site must
be washed before it is removed from the animal; contact of the wash solu-
tion with the cut edge or undersurface of the skin has been shown to
produce artifactual binding of test compound. Liquid detergent designed
for dishwashing is suggested for the wash solution. The animals are killed
and a blood sample collected from the heart or post cava. Residual urine
is collected from the bladder and added to the collected urine. The exposed
skin, selected organs (if part of the experimental design), and the remain-
der of the animal (carcass) are collected and prepared for determination
of the quantity of compound therein. See paragraph (g)(4) of this guideline
for recommendations on organ/tissue collection.
(7) Sample analysis. A total material balance must be obtained for
each animal. Total compound must be determined in each of the following
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samples: Urine, feces, blood (for consistency, it is recommended that blood
mass be assumed equal to 7 percent of body weight (see paragraphs (i)(l)
and (i)(2) of this guideline), wash from the skin, material in or on the
protective device, material remaining in or on the washed skin, material
in selected organs (if collected), and residue in the carcass. Concentration
of the test compound should be determined in the blood and any organ
samples collected.
(f) Data and reporting—(1) Data to be reported. The study report
should include the following information/data derived from the experi-
mental procedure on all animals in all groups:
(i) The method of determination of the limit of sensitivity and the
limit of sensitivity for each sample type in each dose group.
(ii) The actual quantity of test compound administered to each animal
and the mean for each experimental group of four animals.
(iii) Count (disintegrations per minute per gram), quantity of isotope,
quantity of compound and percent of actual dose administered in each
sample for each experimental animal in each experimental group and the
mean of those values for each experimental group of four animals.
(iv) The concentration, quantity, and percent of dose of test compound
in the blood and all tissues analyzed for each animal and the mean of
that value for each experimental group of four animals.
(v) Mass balance totals for each animal in each experimental group
and for the means for each experimental group.
(vi) Determination of the quantity and the percent absorbed for each
animal and for the group (mean). The quantity absorbed is that portion
of the dose which enters the systemic compartment of the organism. The
quantity in/on the skin is localized in the epidermis (mainly the stratum
corneum) and is not available for systemic distribution and toxicity until
it enters the vascular dermis. This determination is based on the following
distribution of the administered dose:
(A) Not absorbed—quantity in skin wash, and on the protective cover.
(B) Absorbable—quantity in/on the washed skin.
(C) Absorbed—quantity in the urine, cage wash, feces, expired air
(if present), blood, organs (if collected), and the remaining carcass.
(2) Final report format and content. The final report should contain
the items that are listed.
(i) Cover page and regulatory documentation. A cover/title page
and additional documentation (i.e., requirements for data submission, good
laboratory practices (GLP) statement and statement of data confidentiality
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claims), if relevant to the study report, must precede the content of the
study format. These requirements are described in 40 CFR parts 158 and
160.
(ii) Table of contents. The table of contents should include a listing
of the elements of the final report such as the summary, an introduction,
the materials and methods, results, discussion, bibliography, tables, figures,
appendices and key subsections as appropriate.
(iii) Body of the report. This item should include such detail that
the reviewer can assess the quality of the study and conformity to the
dermal absorption guidelines or an approved variation of the guidelines.
It should contain the following sections:
(A) Summary. The test report should contain a summary including
a brief description of the study protocol, chemical used, the animals tested,
and the highlights of the results of the study. Any deviations from the
intended protocols should be noted.
(B) Introduction. Include the objectives of the study and the Guideline
reference. The overall experimental design should be explained.
(C) Materials and methods. (7) Test substance. An identification of
the material tested and vehicles used to include the following:
(/) Test material used in test (chemical name, CAS No.)
(//) Properties of the test substance: Chemical structure, form,
radiolabeled, technical label-position, source, radiopurity, lot number,
source, purity, lot number purity, state (liquid or solid), ionization constant
(if applicable), pH (if applicable), solubility in various solvents (if known),
octanol/water partition coefficient (if known)
(///) Vehicles used (if a formulation vehicle is used, it must be identi-
fied but its confidential composition need not be included in the report),
source, lot number.
(2) Test animals. An identification of the experimental animals used
to include the following: Species and strain, sex, source, body weight
range, pretest condition, housing conditions.
(3) Experimental design. The experimental design, as performed in
the study, must be described in complete detail. A step-by-step description
of the entire study in sufficient detail to allow precise understanding of
how the study was performed is required. The description should include,
but not necessarily be limited to, the following: Doses used, number of
animals per dose group, duration of exposure, preparation of the applica-
tion site, area of the application site, dose preparation, dose application,
dose quantitation, method of protecting the application site, urine, feces
and expired air collection, termination method, sample collection methods,
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skin wash, protective device, skin at application site, blood, individual or-
gans (if collected), residual carcass, urine, feces.
(4) Evaluation procedures. A detailed description of the methodology
of the study to include, but not necessarily be limited to, the following:
Method of assignment of animals to test groups, method of verification
of radiopurity, method of determination of actual dose method of sample
collection, storage and analysis, detailed secondary procedures, such as
sample analysis, should be included in the appendices.
(5) Deviation from protocol. Deviations from the protocol or from
an approved variation thereof must be described along with the rationale
for the changes.
(D) Results. This section should provide a narrative summary of the
results of the study. Data generated are best presented in summary tables
and figures included in paragraph (h) of this guideline.
(E) Discussion. (Optional) This section should provide an assessment
of the results of the study, an interpretation of the observations and should
try to explain unexpected findings. The impact of any deviation from the
guideline protocol should be discussed.
(F) Bibliography. (Optional) Complete citations of any documents re-
ferred to in the report. Referenced documents may include previous re-
ports, correspondence to and from the Agency, publications in the tech-
nical literature, and Agency guidelines. Each citation must be sufficiently
complete so as to allow identification and retrieval of the document.
(G) Tables/Figures. These tables and figures should summarize and
illustrate the results of the study by presenting mean values for each exper-
imental group of four animals. Examples are given at the end of this guide-
line.
(H) Appendixes. These should include individual animal data, analyt-
ical methods, results of analysis of the test substance and the dose formula-
tion, protocol, sample calculations and other information as appropriate.
(g) Additional dermal absorption studies. Additional, more specific
studies may be necessary to clarify important points raised in toxicity, me-
tabolism or kinetic studies of a compound. The studies presented below
provide a beginning or outline for designing compound-specific studies
but the individual studies must be designed specifically for the test
compound and the toxicology or kinetic issues of concern. Consultation
with the Agency before performing such a study is strongly recommended.
The individual sample collections/special treatments may, where practical,
be combined with or added to the basic study. Additional studies are as
follows:
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(1) Significant quantity of residue remaining on the washed skin.
(i) For some compounds the washed skin has been found to retain signifi-
cantly more material than was absorbed during the exposure period. In
some cases the difference between absorbed and retained material is suffi-
cient to convert an acceptable risk into an unacceptable risk if all the re-
tained material is considered as absorbed. This study is designed to deter-
mine the fate of that residual material, in particular the portion absorbed
and the rate at which it is absorbed.
(ii) Selected dose levels, as determined from the risk assessment, are
administered to groups of four animals. At 10 h one group from each
dose is terminated as in paragraphs (e)(6) and (e)(7) of this guideline. In
the remaining groups the skin is washed at 10 h, the wash sample collected
for analysis and the groups carried for one or more additional days, up
to 14 days, in metabolism cages. A minimum of 14 days has been sug-
gested by absorption data and a maximum of 21 days has been suggested
by cornified epidermal turnover time in the rat. Suggested exposure dura-
tions, per group of four rats, are 10 h, and 1, 2, 7, 14, and 21 days. Urine
and fecal samples are collected for 24 h intervals. Expired air is collected
if labeled material is expired. At termination samples are collected from
each animal as in paragraphs (e)(6) and (e)(7) of this guideline. Samples
should include any organs/tissues collected in the original study.
(2) Determination of metabolites. Experience has shown that both
qualitative and quantitative production of metabolites of foreign com-
pounds can vary significantly with route of administration. When testing
compounds for which it has been determined that specific metabolites are
of toxicological concern, urine and fecal samples collected in the basic
dermal absorption study may be analyzed for the specific metabolites and
their proportionate production. It may be necessary to dose additional ani-
mals to provide sufficient excreta samples. Blood concentration of label
in dermal absorption studies has shown that compound concentration is
usually too small to allow metabolite identification.
(3) Blood/plasma kinetics study, (i) This study is designed to pro-
vide data to be compared with blood/plasma concentrations at effect and
no-effect doses by the route by which the toxic effect was originally identi-
fied (usually oral). The same species must be used for oral and dermal
determinations. When using these data it is not necessary to obtain and
utilize absorption and excretion parameters in determining MOEs by dif-
ferent routes of administration. Kinetic comparisons of the effect of route
on biological activity are performed by comparing concentrations of active
chemical at the active site following administration by different routes.
This information is extremely hard to obtain, so that obtaining it for any
chemical other than the most important of human drugs is usually imprac-
tical. The most practical surrogate data are blood/plasma concentrations
following different dosing routes. An oral blood/plasma kinetics study
must be performed, using at least the effect and no-effect doses from the
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critical toxicology study and data on blood/plasma concentration with time
as well as volume of distribution and disappearance parameters (half-lives)
obtained before the dermal kinetic study is performed.
(ii) The test compound must be radiolabeled and the radioactivity in
each dose must be sufficient to detect the same minimal concentration
in the blood/plasma. That is, the limit of detection for each dose must
be the same in mass of test material per unit mass of blood/plasma. The
limit of detection must be at least one-hundreth of the maximum blood/
plasma concentration observed following the no-effect oral dose to allow
detection of a hundredfold MOE. A proportionately smaller limit of detec-
tion is necessary for a proportionately larger MOE. If the appropriate limit
of detection cannot be practically obtained the study should not be per-
formed.
(iii) Dermal doses should be selected to bracket the doses (expressed
as milligrams per square centimer) reported for the exposures at risk and
should be at log intervals. Dose preparation, application and protection
of the active site should follow the procedures in the basic dermal absorp-
tion study. The only samples collected for analysis are dosing material
(to determine concentration, homogeneity, and dose applied) and blood
(to determine blood/plasma concentration with dose and time). Analysis
of both whole blood and plasma concentrations is recommended. The rat
has been found to bind many chemicals to the erythrocyte and these data
will allow detection of this process which can have significant effects on
the apparent whole blood kinetics of the bound chemical.
(iv) A minimum of four animals should be used for each dose-dura-
tion-of-exposure data point. Depending upon the size of the blood sample
collected and the time between collections, it may be possible to collect
more than one sample from each animal. However, because of the limited
blood volume in the rat, it is recommended that individual groups of four
rats be used for each exposure (blood sample collection) period.
(v) It is impossible to predict the timing of blood sample collection
following dermal dosing that will be necessary to define the blood/plasma
concentration curve for a particular chemical. Therefore, a preliminary
study is recommended. Use the highest dose proposed for the study, one
or two animals per dose, and collection intervals of 1, 2, 4, 10 and
24 h. Sample collection time can be adjusted from these data to better
fit the expected blood/plasma concentration curve. It must be possible to
identify the peak blood/plasma concentration and characterize the curve
leading to and following it. Note: If blood/plasma concentration is below
the limit of detection for this high dose, it will not be necessary to run
lower doses. No detectable radioactivity can be expected at the lower doses
and the MOE will be derived from the limit of detection following the
high dermal dose.
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(4) Organ/tissue collection, (i) If a target organ for the toxic effect
of the test compound has been identified, it may be useful to determine
test compound concentration, with time and dose, in that tissue following
dermal absorption. Such information can used directly in risk assessment
by comparing the maximum concentration in the target organ following
dermal administration with the maximum concentration following the ef-
fect and no-effect doses by the route used in the critical toxicology tests.
Because of the possibility of saturation of absorption by the dermal route,
it may be impossible to reach a toxic concentration in the target organ
by dermal dosing indicating that there is no risk of concern associated
with dermal exposure.
(ii) Organs should be collected during, as part of, the basic dermal
absorption study. Both concentration and total amount of test material in
the organ (and/or tissue sample) must be obtained; concentration for com-
parison between organs and blood in order to detect evidence of
bioaccumulation in a particular organ and total amount as part of the mate-
rial balance for the dermal absorption study. Organs/tissues suggested for
collection are: The target organs, liver and kidney as metabolic/excretory
organs; heart, lungs, spleen, gonads, adrenals, pancreas, as discrete critical
organs; brain as indicative of the effect of the blood brain barrier; muscle
as a fast storage tissue; and fat as a slow storage tissue.
(5) Volatile compounds, (i) Dermal absorption studies of volatile
compounds can be compromised by inhalation of the vapor, condensation
of the vapor in the metabolism cage with ingestion and condensation of
the vapor on additional areas of the skin. Use of an impermeable cover
on the protective device to counter these effects will produce unrealistic
absorption data. A cover on the protective device consisting of filter paper
impregnated with activated charcoal (or a material such as XAD4
amberlite resin) has been shown to be effective in trapping vaporized or-
ganic test material. A preliminary in vitro test is recommended to deter-
mine the effectiveness of activated charcoal or resin for a particular
compound. For such a test the spacer is glued to a glass plate, a small
measured dose of test material placed within and covered with the charcoal
or resin cover. This model is placed in a metabolism cage maintained at
rat body temperature with a trap on the air outflow to determine whether
any test material escapes the charcoal. After 1 to 2 h the distribution of
the dose is determined from glass plate, spacer, charcoal or resin cover,
metabolism cage wash, and trap. If the dose is confined to the glass plate,
spacer and cover, the study can proceed. If material is found in the cage
wash and/or the trap it may not be possible to perform the study and advice
should be sought from the Agency.
(ii) The experimental design of the dermal absorption study should
follow that of the basic dermal absorption study or an appropriate variation
thereof. A trap may be necessary on the air outflow of the metabolism
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cage if the test compound is excreted by respiration. It should also be
determined if the test material evaporates from the excreta.
(6) Infinite dose studies, (i) This study is designed to address dermal
absorption of the test compound while swimming or bathing, during which
the individual is exposed dermally to a constant concentration of pesticide
from an essentially unlimited source. This is a very tricky study and should
be designed in close consultation with the Agency.
(ii) In this study a reservoir is glued to the skin of the rat, filled
with a water solution of the test compound and covered with an impervious
cover. The exposure area is defined while the dose is varied by varying
the concentration of test compound. Concentrations tested should be at
log intervals and selected to bracket the expected field exposure. Exposure
durations should be chosen to match time in the water for the swimmer
or bather.
(iii) Test compound, test animal, animal preparation, animal process-
ing, and sample analysis should be essentially the same as in the basic
dermal absorption study or an appropriate variation thereof. Results of this
type of study are expressed as flux (mass per unit area per unit time).
(h) Explanatory Documentation. For explanatory documentation of
the Guideline write, Public Docket and Freedom of Information Section,
Field Operations Division, Office of Pesticide Programs, Environmental
Protection Agency, Washington, DC 20460, or call (703) 305-5805. Ask
for OPP-00369 (no charge).
(i) The following references should be consulted for additional back-
ground material on this test guideline.
(1) Laboratory Animal Medicine. Fox, J.G., Cohen, B.J., and Loew,
P.M., eds. Academic (1984).
(2) The Laboratory Rat. Baker, H.J., Lindsey, J.R. and Weisbroth,
S.H., eds. Volume I, Biology and Diseases. Academic, p. 108 (1979).
(3) Zendzian, R.P. Skin Penetration Method Suggested for Environ-
mental Protection Agency Requirements. Journal of the American College
of Toxicology 8:829-835 (1989).
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