United States      Prevention, Pesticides     EPA712-C-96-251
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA    Health Effects Test
          OPPTS 870.8245
          Pharmacokinetics of
          DGBE and DGBA
               '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 870.8245  Dermal pharmacokinetics of DGBE and DGBA.
    (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 the OPPT 40  CFR  795.225 Dermal
Pharmacokinetics of DGBE and DGBA.

    (b) Purpose. The purpose of these studies is to determine the absorp-
tion of diethylene glycol butyl ether (DGBE) after administration by the
dermal route; the biotransformation of DGBE administered dermally;  and
the dermal absorption of DGBE and diethylene glycol butyl ether acetate

    (c) Test procedures—(1) Animal selection—(i) Species. The species
utilized for investigating DGBE and DGBA should be the rat,  a  species
for which historical data on the toxicity and carcinogenicity of many com-
pounds are available and which is used extensively in percutaneous absorp-
tion studies.

    (ii) Animals. Adult female Sprague Dawley rats should be used. The
rats should be 7 to 8 weeks old and weigh 180 to 220 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
isolated 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  DGBE  and  DGBA—(i) Test substances.
These  studies require the use of 14C-labeled  DGBE and DGBA. The use
of 14C-DGBE and  14C-DGBA is required for the determinations in para-
graphs (b) (1), (2), and (3) of this guideline because they will facilitate
the work  and improve the reliability of quantitative determinations.

    (ii) Dosage and treatment. (A) Two  doses of DGBA should be used
in the  study,  a low dose and a high dose. Three doses of DGBE should
be used in the study, a neat low dose, an aqueous low  dose, and neat
high dose. When administered dermally, the high dose level should ideally
induce some overt toxicity such as weight  loss. The low dose level should
correspond to a no-observed-effect-level.

     (B) For dermal treatment, the  doses  should be  applied in 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 micropipette  on a specific area  (for example,
2 cm2) on the freshly shaven skin.

     (iii) Washing  efficiency  study. Before initiation of the  dermal ab-
sorption studies described in paragraph  (c)(2)(iv)(A) of this  guideline, an
initial washing efficiency experiment should be performed  to assess the
extent of removal of the applied DGBE  and DGBA by washing with soap
and water. Groups of four rats should be lightly anesthetized with sodium
pentobarbital. These animals should then be treated with dermal doses of
test substance at the low dose level. Soon after application (5  to  10 min)
the treated animals should be washed  with soap and water then housed
in individual metabolism cages for excreta collection. Urine  and feces
should be collected at 8, 24,  and  48 h following dosing.  Collection of
excreta  should continue  every 24  h if  a  significant amounts  of DGBE,
DGBA, or metabolites continue to be eliminated.

     (iv) Determination of absorption, biotransformation, and excre-
tion. (A) Eight animals should be dosed once dermally with the low dose
of 14C-DGBE.

     (B) Eight animals should  be dosed  once dermally with the high dose
of 14C-DGBE.

     (C) Eight animals should be dosed once dermally with  the low dose
of 14C-DGBA.

     (D) Eight animals should  be dosed  once dermally with the high dose
of 14C-DGBA.

     (E) The high and low doses  of 14C-DGBE and 14C-DGBA should
be kept on  the skin for 24 h. After application,  the animals should be
placed in  metabolism  cages for excreta collection. After 24 h, any test
material remaining  on the  skin  will be washed  off  and  the containment
cell removed. Radiolabeled material in the wash will be  accounted for in
the total recovery. Urine and  feces should be collected at 8, 24, 48, 72,
and 96 h  after dosing, and if  necessary, daily thereafter  until  at least 90
percent of the dose has been excreted or until 7 days after dosing, which-
ever occurs first.

     (3) Observation of animals—(i) Urinary and  fecal excretion. The
quantities  of total 14C excreted in urine and feces by rats dosed as specified
in paragraph (c)(2)(iv) of this guideline should be determined at 8, 24,
48, 72 and  96 h after dosing, and if necessary, daily thereafter until at
least 90 percent of the  dose has been excreted or until 7 days after dosing
(whichever  occurs first). Four animals  from  each group should be used
for this purpose.

     (ii) Biotransformation after dermal dosing. Appropriate qualitative
and  quantitative methods should be  used to assay urine specimens col-
lected  from rats  dosed with DGBE  as specified in paragraph (c)(2)(iv)
of this guideline.  Any metabolite which comprises greater than 10 percent
of the dose should be identified.

     (d) 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-DGBE  and 14C-DGBA.

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

     (vi)  Biotransformation  pathways  and  quantities  of DGBE and
metabolites in urine  collected after  administering single high and low der-
mal doses to rats.