United States      Prevention, Pesticides     EPA712-C-96-252
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.8300
          Dermal Absorption for
          Compounds That Are
          Volatile and Metabolized
          to Carbon Dioxide
               "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 870.8300  Dermal absorption for compounds that are volatile
and metabolized to carbon dioxide.
    (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.  The source material  used in developing this har-
monized OPPTS test guideline is OPPT 40 CFR 795.226 Dermal Absorp-
tion for Compounds That Are Volatile  and Metabolized to Carbon Diox-

    (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 lOOx  the ratio between
total excretion of compound following dermal administration and total ex-
cretion 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 experi-
mental 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 accord-
ance 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 main-
tained 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) Administration of test substance—(i) Test compound. The use
of radioactive test substance is  required for all studies outlined in para-
graph (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 intra-
venously to four rats of each sex  and to  four guinea pigs  of  each  sex.

     (B) Dermal. (7) 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 absorp-
tion  studies described in this guideline, an initial washing efficiency exper-
iment should be conducted to assess the removal of the applied test sub-
stance by  washing the exposed skin area with soap and water and an ap-
propriate  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

     (iii) Dosing  and sampling schedule—(A) Rat  studies—(7) Intra-
venous 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

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 appro-
priate guideline) should be solubilized and assayed for radioactivity to as-
certain if the skin acts as a reservoir for test  substance.

     (B) Guinea pig studies. (7) 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.

     (/) Group  E  should  be  dosed once dermally with the low dose of
test substance.

     (//) Group F should  be  dosed once dermally with the high dose of
test substance.

     (Hi) 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 inci-
dental, 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, in-
cluding 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.

     (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 ad-
ditional background material on this test guideline.

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

     (2)  Jeffcoat, A.R. Research Triangle  Institute. Absorption, distribu-
tion, metabolism and excretion of cyclohexane. Project report No. 5. Re-
search Triangle  Park, N.C. National Institute of Environmental Health
Sciences. Contract NOI-ES-1-5001 (1984).