United States       Prevention, Pesticides      EPA712-C-96-250
          Environmental Protection    and Toxic Substances      June 1996
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.8223
           Pharmacokinetic Test
                 '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 870.8223.  Pharmacokinetic test.
     (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 795.223 Pharmacokinetic

     (b)  Purpose.  The  purpose  of these  tests  is  to  determine  the
bioavailability of a test substance after dermal administration, whether or
not the biotransformation of the test substance is qualitatively and quan-
titatively the same  after dermal and oral  administration, and whether or
not the biotransformation of the test substance is changed qualitatively or
quantitatively by repeated dosing.

     (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 definitions also apply to this test guideline.

     Bioavailability refers to the rate and extent to which the administered
compound is absorbed, i.e. reaches the systemic circulation.

     Relative percent of percutaneous absorption is defined  as  lOOx the
ratio between  total urinary excretion of compound following topical ad-
ministration and total urinary excretion of compound following intravenous

     (d) Test procedures—(1) Animal selection—(i) Species. The species
utilized for  investigating the test substance should be the rat, a species
for which historical data on the toxicity  and carcinogenicity of several
compounds  are available and  which is used extensively in percutaneous
absorption studies.

     (ii) Animals. Adult female Fischer 344 rats should be used. The rats
should be 7 to 9 weeks old and weigh 125  to 175  g. Prior to testing the
animals should be selected at random for each group.  Animals showing
signs of ill health should not be used.

     (iii) Animal care. (A) The animals should be housed in environ-
mentally controlled rooms with 10 to 15 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  rats should be
kept in a quarantine facility for at least 7 days prior to use.

     (B) During the acclimatization period,  the rats should be housed in
cages  on hardwood chip bedding. All animals should  be  provided  with
conventional laboratory diets and water ad  libitum.

     (2) Administration  of test  substance—(i) Test  compound.  Test
studies require the use  of both nonradioactive test substance and 14C-la-
beled test substance. Both  preparations are needed to  investigate under
paragraph (b)(2) of this guideline. The use 14C-test substance is required
to investigate  under  paragraphs (b)(l), (b)(2), and (b)(3) of this guideline
because it will facilitate the work, improve the reliability of quantitative
determinations, and  increase the probability of observing the presence of
previously unidentified metabolities.

     (ii) Dosage and treatment.  (A) Two doses should be used in the
study, a low dose and  a  high dose.  When administered orally,  the  high
dose level should ideally  induce some overt toxicity such as weight  loss.
The low dose level should correspond to a no-effect level.

     (B) The same high and low doses should be administered orally and

     (C) Oral  dosing should be performed by gavage or by administering
encapsulated test substance.

     (D) For dermal treatment, the doses  should be applied  at a volume
adequate to  deliver the  prescribed doses.  The backs of the rats should be
lightly shaved with an electric clipper shortly before treatment. The  dose
should be applied with  a micropipet on 2 cm2 of the freshly  shaven  skin.
The  dosed areas should be  occluded with an aluminum foil patch which
is secured in place with  adhesive tape.

     (iii) Bioavailability study in rats. At least  eight rats should receive
a single intravenous (low) dose of 14C-test substance and serial samples
of blood removed from four animals at 15 min, 30  min,  1 h, 8 h,  24
h, 48 h,  and  96  h.  All animals should be housed  in metabolism cages
and urine and feces collected at 8, 24, 48, 72,  and 96  h. The procedure
should be repeated with eight rats in which  14C-test  substance  is main-
tained in contact with  the  skin for the duration  of the study (96 h). If
dermal absorption cannot be demonstrated, the  study  should be repeated
using a higher dose. Total radioactivity should be measured  in the blood,
urine, and feces samples  collected from all animals.  The results  should
be used to  construct a blood  concentration-time curve and to  calculate
bioavailability by the ratio of the total 96-h urinary excretion of radioactiv-
ity  after dermal  and intravenous  administration.  Bioavailability  is ex-
pressed as (percent dose dermal/percent dose intravenous) x 100 = percent
dermal absorption. Urine  should be saved for metabolite identification, if
it becomes necessary.

     (iv) Biotransformation in rats  after oral  and dermal  administra-
tion. Eight  rats should be  dosed  orally,  and eight  rats should be dosed
dermally (96-h contact) with the high dose of 14C-test substance. The re-
sults of the bioavailability study (see paragraph (d)(2)(iii) of this guideline)
should be evaluated  first to  ensure that the dermal dose applied will result

in the appearance  of radioactivity in the  urine. All  animals should be
housed in metabolism cages allowing for separate collection of urine and
feces at 8, 24, 48, 72, and 96 h. The parent compound and any metabolite
that  comprises greater than 10 percent of the dose should be identified
in the urine. These results should be qualitatively compared to the urinary
excretion data obtained in the low dose bioavailability study (see paragraph
(d)(2)(iii) of this guideline); metabolites in the low dose urine should also
be identified if a different pattern of metabolism is evident.

     (v) Repeated dosing study. Four rats should receive a series of single
daily oral doses of nonradioactive  test substance over  a period of at least
14 days, followed at 24 h  after the last dose by  a single oral dose of
14C-test substance. Each dose should be at the low-dose level. If the pat-
tern  of urinary metabolite excretion is qualitatively different from that ob-
tained with  the  orally dosed animals in the  single-dose biotransformation
study  at  24  and  48  h  (see  paragraph  (d)(2)(iv) of  this  guideline),
metabolites  should be identified in accordance with the procedure given
in paragraph (d)(2)(iii) of this guideline.

     (vi) Skin washing study. If greater than  10 percent of test substance
is absorbed through  the skin (see  paragraphs (d)(2)(ii) and (d)(2)(iii) of
this  guideline) then a washing  efficacy experiment  should be performed
to assess the extent of removal of the applied test  substance  by washing
with soap and water. Four  rats  should be lightly anesthetized and  treated
with a dermal dose of test compound previously shown to result in measur-
able  percutaneous absorption greater than 10 percent.  Soon after applica-
tion  (5 to 10  min) the treated animals should be washed with soap and
water, then  housed in individual metabolism cages  for excreta collection.
Measurements of total radioactivity in urine  and feces should be made
in the same manner as described in paragraph (d)(2)(iii) of this guideline.

     (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, strain, and supplier of laboratory animals.

     (ii) Information  on the  degree (i.e., specific activity for a radiolabel)
and sites of labeling of the test substances.

     (iii) A  full description of the sensitivity  and precision of all  proce-
dures used to produce the data.

     (iv) Relative percent absorption by the dermal route for rats adminis-
tered low and high doses of 14C-test substance, compared with 100 percent
of the intravenous dose.

     (v) Quantity of isotope, together with percent recovery of the adminis-
tered dose, in feces, urine, and blood.

     (vi) Biotransformation pathways and quantities of the test substance
and metabolites in urine collected after administering single high and low
oral and dermal doses.

     (vii) Biotransformation pathways and quantities of test substance and
metabolites in  urine  collected after administering  repeated  low doses of
test substance to rats.