United States       Prevention, Pesticides      EPA712-C-96-197
           Environmental Protection    and Toxic Substances      June 1996
           Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.2600
           Skin Sensitization
                 '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 870.2600   Skin sensitization.
     (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 798.4100 Dermal Sen-
sitization; OPP 81-6  Dermal Sensitization (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation;  Human and Domestic Animals)
EPA report 540/09-82-025, 1982; and OECD 406 Skin Sensitization.

     (b) Purpose. Determination of the potential to cause or elicit skin-
sensitization reactions (allergic contact dermatitis) is an important element
in evaluating a  substance's  toxicity. Information derived from skin-sen-
sitization tests serves to identify possible hazards to a population exposed
repeatedly to a test  substance. The test selected should identify  substances
with significant  allergenic potential and minimize false  negative  results.

     (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.

     Challenge exposure is an experimental exposure of a previously treat-
ed subject to a test  substance following an induction period, to determine
if the subject will react in a hypersensitive manner.

     Induction exposure is an experimental exposure of a subject to a test
substance with the intention of inducing a hypersensitive state.

     Induction period is a period of a  least 1  week following an induction
exposure during which a hypersensitive state may develop.

     Skin sensitization  (allergic contact  dermatitis) is  an  immunologically
mediated cutaneous reaction to a substance.  In the human, the responses
may be characterized by pruritis, erythema,  edema, papules, vesicles,
bullae, or a combination of these. In other  species, the  reactions  may differ
and only erythema and edema may be seen.

     (d) Principle of the test method. Following initial exposure to a test
substance,  the  animals are subjected,  after a  period of not less  than
1 week, to a challenge  exposure with the test  substance to establish wheth-
er a hypersensitive  state has been induced. Sensitization  is determined by
examining the reaction to the challenge exposure and comparing this reac-
tion with that of the initial induction exposure.  The test animals are ini-
tially exposed to the test substance by intradermal and/or epidermal appli-
cation (induction exposure).  Following a rest period of 10 to 14 days (the
induction period),  during  which an immune response may develop, the
animals are exposed to a challenge dose. The  extent and degree  of skin

reaction to the challenge exposure is compared with that demonstrated by
control animals  that undergo sham treatment during  induction and then
receive the challenge exposure.

     (e) Test procedures. (1) Any of the following test methods is consid-
ered to be acceptable.

     (i) Buehler test.

     (ii) Guinea-pig maximization test (GPMT).

     (iii) Other.

     (A) Open epicutaneous test.

     (B) Maurer optimization test.

     (C) Split adjuvant technique.

     (D) Freund's complete adjuvant test.

     (E) Draize sensitization test.

     (2) The GPMT of Magnusson and Kligman, which uses adjuvant, and
the nonadjuvant Buehler test are given preference  over other methods. Al-
though strong preference is given to either the Buehler test or the GPMT,
it is recognized  that other  tests may give useful results. If other tests are
used, the tester should provide justification/reasoning  for their use,  meth-
ods and protocols must be provided, and each test should include a positive
and a negative control group.

     (f)  Screening tests. The mouse ear swelling test (MEST) (see para-
graphs (i)(9), (i)(10), (i)(ll), and  (i)(12)  of this guideline) or the  local
(auricular) lymph node  assay (LLNA)  (see paragraphs  (i)(13), (i)(14),
(i)(15), and (i)(16) of this guideline) in the mouse may be used as screen-
ing tests to detect moderate to strong sensitizers. If a  positive result is
seen in either assay, the test substance may be designated  a potential sen-
sitizer, and it may not be necessary to  conduct  a further test  in guinea
pigs.  If the LLNA or MEST does not indicate sensitization, the test sub-
stance should not be designated a nonsensitizer without confirmation in
an accepted test using guinea pigs.

     (g) Animal selection—(1) Species and strain. The young adult guin-
ea pig is preferred. Commonly used laboratory strains should be employed.
If other species  are used, the tester should provide justification/reasoning
for their selection.

     (2) Housing and feeding. The temperature  of the experimental ani-
mal room  should be 20 + 3 °C with the  relative humidity 30-70 percent.
Where the lighting is artificial,  the sequence   should  be  12 h  light/
12 h  dark. Conventional laboratory diets may be used with an unlimited

supply of drinking water. It is essential that guinea pigs receive an ade-
quate amount of ascorbic acid.

     (3) Number and sex. The number and sex will depend on the method
chosen. Either  sex may be used in the  Buehler test and  the GPMT. If
females are used,  they should be nulliparous and not pregnant. The Buehler
test recommends  using a minimum of 20 animals in the treatment  and
at least 10 as controls. At least  10 animals in the treatment group  and
5 in the control group should be used with the GPMT, with the stipulation
that if it is not possible to conclude that the  test substance is a sensitizer
after using fewer than 20 test and  10  control guinea pigs, the testing of
additional animals to give a total of at least 20 test and 10 control animals
is strongly recommended.

     (4) Control  animals, (i) The sensitivity and reliability of the experi-
mental technique  used should be assessed every 6 months in naive animals
by the use of positive control substances known to have mild-to-moderate
skin-sensitizing properties. In a properly conducted test, a response of at
least 30 percent in an adjuvant test and at least 15 percent in a nonadjuvant
test should be expected for mild-moderate sensitizers. Preferred substances
are hexylcinnamic aldehyde (CAS No.  101-86-0), mercaptobenzothiazole
(CAS No. 149-30-4), and benzocaine (CAS No.  94-09-7). There may
be  circumstances where, given adequate justification,  other  control sub-
stances meeting the above criteria  may be used.

     (ii) Depending upon the test selected, animals may be used as their
own controls, but usually there will be a separate group of  sham-treated
animals that are exposed to the test substance only after the induction pe-
riod, whose reactions are compared to those  of the animals that have re-
ceived both induction and challenge  exposures.

     (5) Dose  levels. The dose level will depend on the test method se-
lected. In the Buehler test, the concentration  of the  induction dose should
be high enough to  cause mild irritation,  and the challenge  dose should
use the highest non-irritating concentration. In the GPMT, the concentra-
tion of the induction dose should be well tolerated systemically, and should
be high enough to cause mild-to-moderate skin irritation; the  GPMT chal-
lenge dose should use the highest non-irritating concentration.

     (6) Observation of animals, (i) Skin reactions should be graded  and
recorded after the challenge exposures at the time specified by the meth-
odology selected. This  is usually at  24, 48, and 72 h. Additional notations
should be made as necessary to fully describe  unusual responses.

     (ii) Regardless of the test method selected, initial and terminal body
weights should be taken and recorded.

     (7) Procedures. The procedures to be used are those described by
the test method chosen. Brief summaries  are given here, but the  tester

should refer to the original literature for more complete guidance on con-
ducting the Buehler test (under paragraphs  (i)(l), (i)(2), (i)(3), and (i)(4)
of this guideline) or the GPMT (under paragraphs (i)(5), (i)(6), (i)(7), and
(i)(8) of this guideline).

     (i) The Buehler test uses topical administration  via  a  closed patch
on days 0, 6-8,  and 13-15  for induction,  with topical challenge  of the
untreated flank for 6 h on day 27-28. Readings are made approximately
21 and 24 h after removing the challenge patch, and again 24 h after that.
If the  results  are equivocal,  the animals may be rechallenged one week
later, using either the original control group or  a new control group for
comparison. See paragraphs (i)(l), (i)(2), (i)(3), and (i)(4) of this guideline.

     (ii)  The GPMT uses intradermal injection with and without Freund's
complete adjuvant (FCA) for induction,  followed on days 5-8 by topical
irritation/induction, followed  by topical challenge for 24 hours on day 20-
22. Readings are made approximately 24 h after removal of the challenge
dose, and again after another 24 h. As with the Buehler test,  if the  results
are equivocal, the animals may be  rechallenged  1 week later. If only 10
animals were used initially and gave equivocal results, the use of an addi-
tional  10 experimental  and  5 control  animals  is  strongly recommended.
See paragraphs (i)(5), (i)(6), (i)(7), and (i)(8) of this guideline.

     (iii) Blind reading  of both test and  control  animals is recommended.

     (iv) Removal of the test material should be accomplished with water
or an appropriate solvent, without altering the existing response or the in-
tegrity of the epidermis.

     (v) Hair is removed from the site of application by clipping,  shaving,
or possibly by depilation, depending on the test selected.

     (h) Data  and reporting. Data should be summarized in tabular form,
showing for each individual animal the skin reaction, results of the  induc-
tion  exposure, and the challenge exposure at times indicated by the method
chosen. As a  minimum, the  erythema and  edema should be graded and
any unusual finding should be recorded.

     (1) Evaluation of the results. The  evaluation of  results will provide
information  on the proportion of each group that became sensitized and
the extent (slight, moderate,  severe) of the sensitization reaction in each
individual animal.

     (2) Test report. In addition to the reporting requirements as specified
under  40 CFR part 158 (for pesticides) and 40 CFR part  792, subpart
J (for toxic  substances), the  following specific information should  be re-

     (i) A description of the method used and  the commonly accepted

     (ii) Information on the positive control study, including the positive
control substance used, the method used, and the time conducted.

     (iii) The number,  species, strain,  age, and sex of the test animals.

     (iv) Individual weights of the animals at the start of the test and at
the conclusion of the test.

     (v) A brief description of the grading system.

     (vi) Each reading made on each individual animal.

     (vii) The chemical identification and relevant physicochemical prop-
erties of the test substance.

     (viii) The vehicles used for induction and challenge, and justification
for their use, if other than water or physiological saline. Any material that
might reasonably be expected to react with or  enhance  or retard absorption
of the test substance should be reported.

     (ix) The total amount of test substance applied for induction and chal-
lenge, and the technique of application in each case.

     (x) The source from which the animals were obtained, housing condi-
tions, diet,  evidence for adequate ascorbic acid,  and  any  other information
on animal care that may be relevant.

     (xi) Histopathological findings, if any.

     (xii) Discussion of results.

     (i) References. The following references should be consulted for ad-
ditional background information on this test guideline.

     (1) Buehler, E.V.  Delayed contact hypersensitivity in the guinea pig,
Archives of Dermatology 91: 171-177 (1965).

     (2) Ritz, H.L. and Buehler, E.V. Planning,  conduct and interpretation
of guinea pig sensitization patch tests in Current Concepts in Cutaneous
Toxicity, ed. V. Drill and P. Lazar. Academic,  New  York, NY. pp. 25-
42 (1980).

     (3) Buehler, E.V.  Occlusive patch method for skin  sensitization in
guinea pigs: the Buehler  method. Food and Chemical Toxicology 32:97-
101 (1994).

     (4)   Buehler,   E.V.   Prospective  testing  for   delayed   contact
hypersensitivity in  guinea pigs: the  Buehler  method; in  Methods  in
Immunotoxicology,  Vol. 2, ed. G. Burleson, A.  Munson, and J.  Dean.
Wiley, NY, pp. 343-356 (1995).

     (5) Magnusson, B. and Kligman, A.M. The identification of contact
allergens by animal assay. The guinea pig maximization test. Journal of
Investigative Dermatology 52: 268-276 (1969).

     (6) Magnusson, B. and Kligman, A.M. Allergic contact dermatitis in
the guinea pig. Charles C. Thomas, Springfield, IL (1970).

     (7) Magnusson,  B. Identification of contact  sensitizers by animal
assay. Contact Dermatology 6:46 (1980).

     (8) Magnusson, B. et al. Determination of skin sensitization potential
of chemicals. Predictive testing in guinea pigs. Arbete och Hdlsa: 26(E)

     (9) Gad, S.C. et al. Development and validation of an alternative der-
mal  sensitization test: the mouse  ear  swelling test (MEST).  Toxicology
and Applied Pharmacology 84: 93-114 (1986).

     (10) Maisey, J. and Miller, K., Assessment of the ability of mice fed
on Vitamin-A supplemented diet to respond to a variety of potential  con-
tact sensitizers. Contact Dermatitis 15: 17-23 (1986).

     (11) Thorne, P.S. et al., The noninvasive mouse ear swelling assay.
I. Refinements for detecting weak  contact sensitizers. Fundamental and
Applied Toxicology 17:790-806 (1991).

     (12) Thorne, P.S. et al. The noninvasive mouse ear swelling assay.
II. Testing the contact sensitizing potency of fragrances. Fundamental and
Applied Toxicology 17:807-820 (1991).

     (13) Kimber, I. et al. The murine local lymph  node  assay for identi-
fication of contact allergens: a preliminary evaluation of in situ measure-
ment of lymphocyte proliferation. Contact Dermatitis 21:215-220 (1989).

     (14) Kimber, I. et al. Identification of contact allergens using the mu-
rine local lymph node assay: comparisons with the Buehler Occluded Patch
Test in guinea pigs. Journal of Applied  Toxicology 10:  173-180 (1990).

     (15) Kimber, I.  et al.  The murine  local lymph node assay: results
of an interlaboratory trial. Toxicology Letters 55:203-213 (1991).

     (16) Basketter,  D.A.  et al.  Interlaboratory evaluation of the  local
lymph node assay with 25 chemicals and comparison with guinea pig test
data. Toxicology Methods 1:30-43 (1991).