United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–252

June 1996

Health Effects Test

Guidelines

OPPTS 870.8300

Dermal Absorption for

Compounds That Are

Volatile and Metabolized

to Carbon Dioxide

‘‘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 870.8300 Dermal absorption for compounds that are volatile

and metabolized to carbon dioxide.

(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. The source material used in developing this harmonized OPPTS test guideline is OPPT 40 CFR 795.226 Dermal Absorption for Compounds That Are Volatile and Metabolized to Carbon Dioxide.

(b) Purpose. The purpose of these studies is to determine the extent

of absorption of test substance after dermal administration.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR

Part 792—Good Laboratory Practice Standards (GLP) apply to this test

guideline. The following definition also applies to this test guideline.

Percent of dermal absorption is defined as 100× the ratio between

total excretion of compound following dermal administration and total excretion following intravenous administration.

(d) Test procedures—(1) Animal selection—(i) Species. The rat and

guinea pig should be used.

(ii) Test animals. Adult male and female Fischer 344 rats should

be used. At 7 to 9 weeks of age, the male rats should weigh 125 to 175

g and the female rats 110 to 150 g. Guinea pigs, 5 to 7 weeks old, should

also be used. The animals should be purchased from a reputable dealer

and should be identified upon arrival. The animals should be selected at

random for the testing groups and any animal showing signs of ill health

should not be used. In all studies, unless otherwise specified, each experimental group should contain at least four animals of each sex for a total

of at least eight animals.

(iii) Animal care. (A) Animal care and housing should be in accordance with DHEW Publication No. (NIH)–85–23 (1985), Guidelines for the

Care and Use of Laboratory Animals.

(B) The animals should be housed in environmentally controlled

rooms with at least 10 air changes per hour. The rooms should be maintained at a temperature of 25 ± 2 °C and humidity of 50 ± 10 percent with

a 12–h light/dark cycle per day. The animals should be kept in a quarantine

facility for at least 7 days prior to use.

(C) During the acclimatization period, the animals should be housed

in suitable cages. All animals should be provided with certified feed and

tap water ad libitum. The guinea pig diet should be supplemented with

adequate amounts of ascorbic acid in the drinking water.
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(2) Administration of test substance—(i) Test compound. The use

of radioactive test substance is required for all studies outlined in paragraph (d)(2)(ii) of this guideline. Ideally, the purity of both radioactive

and nonradioactive test substance should be greater than 99 percent. If

the purity is less than 99 percent or if the radioactive and nonradioactive

compounds differ significantly, the EPA should be consulted.

(ii) Dosage and treatment—(A) Intravenous. The low dermal dose

of test substance (in an appropriate vehicle) should be administered intravenously to four rats of each sex and to four guinea pigs of each sex.

(B) Dermal. (1) For dermal treatment, the low and high doses should

be dissolved in a suitable vehicle and applied at a volume adequate to

deliver the doses. The backs of the animals should be lightly shaved with

an electric clipper 24 h before treatment. The chemical should be applied

to the intact shaven skin (approximately 2 cm2 for rats, 5 cm2 for guinea

pigs). To prevent losses of test substance by evaporation, the use of a

stainless skin depot glued to the backs of the animals with a cyanoacrylate

adhesive is recommended. The depot is fitted with a screw cap to prevent

the escape of vapor and with a basket of activated charcoal to adsorb

compound which vaporizes (see paragraph ()() of this guidelineRef. 1).

(2) Washing efficiency study. Before initiation of the dermal absorption studies described in this guideline, an initial washing efficiency experiment should be conducted to assess the removal of the applied test substance by washing the exposed skin area with soap and water and an appropriate solvent. The low dose of test substance should be applied to

four rats and four guinea pigs. After application (2 to 5 min), the areas

should be washed with soap and water and an appropriate solvent (two

rats, two guinea pigs). The amounts recovered in the washings should be

determined to assess efficacy of test substance removal by washing the

skin.

(iii) Dosing and sampling schedule—(A) Rat studies—(1) Intravenous study. Group A is to be dosed once intravenously at the low dose

of test substance. The rats should be placed in individual metabolic units

(see paragraph ()() of this guidelineRef. 2) for collection of urine, feces

and expired air at 8, 24, 48, 72 and 96 h after dosing. The metabolic

units are to be cleaned after the final collection period to remove any

excreta that might adhere to the units.

(2) Dermal studies. The test substance should be kept on the skin

for a minimum of 6 h. Group B should be dosed once dermally with the

low dose of test substance. Group C should be dosed once dermally with

the high dose of test substance. During and after the exposure period, each

animal should be placed in an individual metabolic unit for the collection

of urine, feces and expired air at 8, 24, 48, 72 and 96 h after dosing.

The metabolic units are to be cleaned after the final collection period to
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remove any excreta that might adhere to the units. At the time of removal

of the skin depot, the treated area and the steel depot should be washed

with an appropriate solvent to remove any test substance. Also, the test

substance should be desorbed from the activated charcoal with a suitable

solvent. The washings and extracts should be assayed to recover residual

test substance. At the termination of the experiments, each animal should

be sacrificed and the exposed skin area removed. The skin (or an appropriate guideline) should be solubilized and assayed for radioactivity to ascertain if the skin acts as a reservoir for test substance.

(B) Guinea pig studies. (1) Intravenous study. Group D is to be

dosed once intravenously at the low dose of test substance. The guinea

pigs should be placed in individual metabolic units for the collection of

urine, feces and expired air as the Group A.

(2) Dermal studies. The studies conducted on groups B and C as

specified in paragraph (d)(2)(iii)(A)(2) of this guideline should be repeated

using four guinea pigs per group.

(i) Group E should be dosed once dermally with the low dose of

test substance.

(ii) Group F should be dosed once dermally with the high dose of

test substance.

(iii) Measurements-excretion. The quantities of radioactivity excreted

in the urine, feces, and expired air should be determined at appropriate

time intervals as described above and, if necessary, daily thereafter until

at least 90 percent of the applied dose has been excreted or until 7 days

after dosing (whichever occurs first).

(e) Data and reporting—(1) Treatment of results. Data should be

presented in tabular form.

(2) Evaluation of results. All observed results, quantitative or incidental, should be evaluated by an appropriate statistical method.

(3) Test report. In addition to the reporting requirements as specified

in 40 CFR part 792, subpart J, the following specific information should

be reported:

(i) Species and strains of laboratory animals.

(ii) Information on the sites and extent of test substance labeling, including specific activity, chemical purity, radiochemical purity, and results

of chromatography.

(iii) A full description of the sensitivity, precision, and accuracy of

all procedures used to produce the data.
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(iv) Percent of absorption of test substance after dermal exposures

to rats and guinea pigs.

(v) Quantity and percent recovery of test substance in feces, urine,

and expired air. In dermal studies on rats and guinea pigs, include recovery

data for skin, skin washings, and residual compound in the skin depot

and charcoal.

(f) References. The following references should be consulted for additional background material on this test guideline.

(1) Susten, A.S. et al In vivo percutaneous absorption studies of volatile solvents in hairless mice. I. Description of skin-depot. Journal of Applied Toxicology 6:43–46 (1986).

(2) Jeffcoat, A.R. Research Triangle Institute. Absorption, distribution, metabolism and excretion of cyclohexane. Project report No. 5. Research Triangle Park, N.C. National Institute of Environmental Health

Sciences. Contract NOI–ES–1–5001 (1984).