.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–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’’

### i 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 theNational 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 proceduresthat 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 theEPA 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.’’

### 1 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 betweentotal 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 healthshould 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 controlledrooms 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.

### 2 (2) Administration of test substance—(i) Test compound . The useof 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 doseof 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 cm 2 for rats, 5 cm 2 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

### 3 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 shouldbe 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.

### 4 (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).