United States       Prevention, Pesticides     EPA712-C-96-350
          Environmental Protection    and Toxic Substances     June 1996
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.7600
           Dermal Penetration
                 'Public Draft"

     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

     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:

     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,  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

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.

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

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.

     (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

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

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

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

     (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,

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-

     (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


     (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

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-

     (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.


     (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


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).