United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–350

June 1996

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 Pesticide 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 biological 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 substitute 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 specific 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 whether 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 dermal 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 determined 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 compared 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 position which is part of the ‘‘core’’ of the compound. The label should follow

the compound and its major metabolites until excreted. To the extent practical, 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, 36Cl, 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/chemical test for identifying the compound is used. The specificity and sensitivity 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 material used in the commercial formulation (formulation blank). Dilutions are

made with the field vehicle, usually water, to produce a solution or suspension. 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 special 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 depends upon how small a quantity of the test compound is considered necessary 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 nominal 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 pesticide 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 experimental unit. Studies using fewer exposure durations will use fewer animals 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 absorption 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. Compounds 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 maximum 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 μL/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-resistant) 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
5

be used but care must be taken to rinse the area throughly to remove residual 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 showing

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 spreading. The material is spread evenly until a film is formed over the application 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 determining 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 rectangle, 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/detergent 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 solution 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 remainder 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)(1)

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 experimental 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
7

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 e1ements 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. (1) Test substance. An identification of

the material tested and vehicles used to include the following:

(i) Test material used in test (chemical name, CAS No.)

(ii) 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)

(iii) Vehicles used (if a formulation vehicle is used, it must be identified 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 application 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,
8

skin wash, protective device, skin at application site, blood, individual organs (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 referred to in the report. Referenced documents may include previous reports, correspondence to and from the Agency, publications in the technical 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 experimental group of four animals. Examples are given at the end of this guideline.

(H) Appendixes. These should include individual animal data, analytical methods, results of analysis of the test substance and the dose formulation, 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, metabolism 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 significantly more material than was absorbed during the exposure period. In

some cases the difference between absorbed and retained material is sufficient to convert an acceptable risk into an unacceptable risk if all the retained material is considered as absorbed. This study is designed to determine 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 suggested by absorption data and a maximum of 21 days has been suggested

by cornified epidermal turnover time in the rat. Suggested exposure durations, 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 compounds 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 animals 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 provide data to be compared with blood/plasma concentrations at effect and

no-effect doses by the route by which the toxic effect was originally identified (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 different 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 impractical. 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
10

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 detection is necessary for a proportionately larger MOE. If the appropriate limit

of detection cannot be practically obtained the study should not be performed.

(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 absorption 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-duration-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 effect 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 comparison between organs and blood in order to detect evidence of

bioaccumulation in a particular organ and total amount as part of the material 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 organic test material. A preliminary in vitro test is recommended to determine 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
12

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 processing, 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 background material on this test guideline.

(1) Laboratory Animal Medicine. Fox, J.G., Cohen, B.J., and Loew,

F.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 Environmental Protection Agency Requirements. Journal of the American College

of Toxicology 8:829–835 (1989).