United States       Prevention, Pesticides     EPA712-C-96-196
           Environmental Protection    and Toxic Substances     June 1996
           Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.2500
           Acute Dermal Irritation
                 '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 870.2500  Acute dermal irritation.
     (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 materials used in developing this har-
monized OPPTS test guideline are 40 CFR 798.4470 Primary Dermal Irri-
tation; OPP 81-5 Primary Dermal Irritation (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation; Human and Domestic Animals)
EPA report 540/09-82-025, 1982; and OECD 404 Acute Dermal Irritation/
Corrosion .

     (b) Purpose. Determination of the irritant and/or  corrosive effects on
skin of mammals is useful in the assessment and evaluation  of the toxic
characteristics of a substance where  exposure by the dermal route is likely.
Information derived from this test serves to indicate the existence of pos-
sible hazards likely to arise from exposure of the skin to the test substance.
Data  on primary dermal irritation  are required  by 40  CFR  part 158  to
support the registration of each manufacturing-use product and end-use
product. See specifically  § § 158.50 and  158.340  to determine whether
these data must be submitted.

     (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.

     Dermal corrosion is the production of irreversible tissue damage  in
the skin following the application of the test substance.

     Dermal irritation  is  the  production  of  reversible inflammatory
changes in the skin following  the application of a test substance.

     Pharmacological effect means  any chemically  induced physiological
changes in the test animal.

     Target organ means any organ of a test animal showing evidence
of an effect of chemical treatment.

     (d) Principle of the test  methods. (1) The substance to be tested
is  applied in a single dose to the  skin of several  experimental animals,
each animal serving as its own control (except when  severe irritation/corro-
sion is suspected and  the  stepwise  procedure  is used  (see  paragraph
(f)(l)(iii) of this guideline)).  The degree of irritation is read  and  scored
at  specified intervals and is further described to provide a complete evalua-
tion of the effects. The duration of the study should be sufficient to permit
a full evaluation of the reversibility or irreversibility of the  effects ob-
served but need not exceed 14 days.

     (2) When testing  solids (which may be pulverized if considered nec-
essary), the test substance should be moistened sufficiently with water or,
where necessary, a suitable vehicle, to ensure good contact with the skin.
When vehicles are used, the influence of the vehicle on  irritation of skin
by the test substance should be taken into account. Liquid test substances
are generally used undiluted.

     (e) Initial considerations. (1) Strongly acidic or alkaline substances,
for example with a demonstrated pH of 2 or less, or 11.5 or greater, need
not be tested for primary dermal irritation, owing to their predictable  corro-
sive properties.

     (2) It is unnecessary to test materials which have been  shown to be
highly toxic  (LD50 less than  200 mg/kg) by  the dermal  route or have
been  shown  not to produce irritation of the skin at the limit  test dose
level of 2000 mg/kg body weight.

     (3) It may not be  necessary to test in vivo  materials  for which  corro-
sive  properties are predicted on the basis of results from  well  validated
and accepted in vitro  tests. If  an in vitro test is performed before  the in
vivo test, a description or  reference to  the test,  including details of the
procedure, must be given together with results  obtained with the test and
reference  substances.

     (4) It may not be necessary to test materials for which corrosive po-
tential is predicted from structure-activity relationships.

     (f) Test procedures—(1)  Animal selection—(i) Species and strain.
The  albino  rabbit  is recommended as the  preferred species. If another
mammalian species is  used, the tester should provide justification/reason-
ing for its selection.

     (ii) Number of animals.  At least  six healthy adult animals should
be used unless justification/reasoning for  using fewer animals is provided.
It is recommended that a stepwise procedure be used to expose one animal,
followed by additional  animals  to clarify equivocal responses.

     (iii) Stepwise exposure of animals. A single rabbit may be used if
it is suspected that the  test material might produce severe  corrosion.  Three
test patches  are applied concurrently or  sequentially to  the  animal. The
first patch is removed  after 3 min. If no  serious skin reaction is observed,
the second patch is removed after 1 h. If observations indicate that expo-
sure  can be continued  humanely, the third patch is removed after 4  h and
the responses graded.  If a corrosive effect  is observed after  either  3 min
or 1  h of exposure, the test is  immediately terminated by removal  of the
remaining patches. If  a corrosive  effect  is  observed after an exposure of
up to 4 hours, then further animal testing is not required. If no  corrosive
effect is observed in one animal after a 4-h exposure, the  test is completed
using two additional animals, each with  one patch  only, for an exposure

period of 4 h. If it is expected that the test substance will not produce
severe irritancy or corrosion, the test may be started using three animals,
each receiving one patch for an exposure period of 4 h.

     (2) Control  animals. Separate animals are not recommended for an
untreated  control group.  Adjacent areas of untreated skin of each animal
may serve as a control for the test.

     (3) Dose level. A  dose of 0.5 mL of liquid  or 500 mg of solid or
semisolid is applied to the test site.

     (4) Preparation of test  area. Approximately 24 h before the test,
fur should be  removed  from the  test area by clipping  or  shaving  from
the dorsal area of the trunk of the animals. Care  should be taken to avoid
abrading the skin. Only  animals with healthy intact skin should be used.

     (5) Application of the test substance, (i) The recommended expo-
sure duration is normally 4 h. unless corrosion is observed (see paragraph
(f)(l)(iii)  of this guideline). Longer exposure may be indicated under cer-
tain conditions (e.g. expected  pattern of human use and exposure). At the
end of the  exposure period, residual test substance should generally  be
removed,  where practicable, using  water or an appropriate solvent, without
altering the existing response or the integrity of the epidermis.

     (ii) Purity/grade for pesticides. A manufacturing-use product should
be  tested  to support the registration of a  manufacturing-use product and
an end-use product should be tested to support the registration of an end-
use product. The  lot of the substance tested should be the  same throughout
the duration of the  study, and the  research sample  should be stored under
conditions that maintain its purity and stability. The composition of each
lot  of the test substance should be determined,  including the name and
quantities of known contaminants and impurities,  as far  as is technically
feasible.  The  determination should  include quantities of unknown mate-
rials,  if any, so that 100 percent of the test sample is accounted for. The
test substance should be within the limits, if any  certified in accordance
with OPPTS 830.1750. When vehicles are used, the influence of the vehi-
cle on irritation of skin by the test substance should by taken into account.
If a vehicle is used, it should not alter the absorption, distribution, metabo-
lism,  retention or the chemical properties of the test substance nor should
it enhance, reduce, or alter its toxic characteristics. At the levels used in
the study, the  vehicle should not produce physiological effects, and should
resemble the vehicle to be used under expected conditions of use. It should
not interfere with the nutritional status of the animals, nor produce physio-
logical effects. If the test  substance is to  be incorporated into a vehicle,
the period during which the  test substance is  stable in  such a mixture
should be determined prior to the start of the study. Alternatively, deter-
mination  of the  stability of the test or control  substance in statistically
randomized samples of vehicle mixture should be made periodically during

      the study  (see  40 CFR  Part  160—Good Laboratory Practice Standards
      (GLPS)). If the test substance is  incorporated  into a vehicle, its homo-
      geneity and concentration should  be  determined prior to the  start of the
      study and periodically during the study.

           (iii) The test substance should be applied  to  a small  area (approxi-
      mately 6 cm2)  of skin and covered  with a gauze patch,  which is held
      in place with nonirritating tape. In the case of liquids or some pastes,
      it may be necessary to apply the test  substance to the gauze patch and
      apply  that to the skin. The patch  should be loosely held in contact with
      the skin by means  of a  suitable semiocclusive  dressing for the duration
      of the exposure period. Access  by the animal to the patch and resultant
      ingestion/inhalation of the test substance should be prevented.

           (6) Observation period. The duration of the observation period need
      not be rigidly fixed. It should be sufficient to fully  evaluate the reversibil-
      ity or  irreversibility of the effects  observed. It  need not exceed 14 days
      after application.

           (7) Clinical examination and scoring, (i) After removal of the patch,
      animals should be  examined for  signs of erythema and edema and the
      responses  scored within  30-60 min, and at 24, 48, and 72 h after patch

           (ii) Dermal irritation should be scored and  recorded according to the
      grades in the following Table. 1. Further observations may be needed, as
      necessary, to establish reversibility. In addition to the observation of irrita-
      tion, any lesions and other toxic effects should be fully described.
                               Evaluation of Skin Reaction
Erythema and Eschar Formation:
  No erythema
  Very slight erythema (barely perceptible)
  Well-defined erythema
  Moderate to severe erythema	
  Severe erythema (beet redness) to slight eschar formation (injuries in depth)
  Maximum possible  	
Edema Formation:
  No edema
  Very slight edema (barely perceptible) 	
  Slight edema (edges of area well defined by definite raising) 	
  Moderate edema (raised approximately 1 mm) 	
  Severe edema (raised more than 1  mm and extending beyond area of exposure
  Maximum possible  	

           (g)  Data and reporting—(1) Data summary. Data should be sum-
      marized  in tabular form,  showing for each individual animal the irritation
      scores for erythema  and edema at 30 to 60  min, and 24, 48, and 72 h
      after  patch removal,  any lesions, a  description of the  degree and  nature

of irritation,  corrosion  and reversibility,  and any  other toxic effects ob-

     (2) Evaluation of results. The dermal irritation scores should be eval-
uated in conjunction with the nature and reversibility or otherwise of the
responses observed. The individual  scores do  not represent an  absolute
standard for  the irritant properties of a material.  They should be viewed
as reference  values which are  only meaningful when supported by a full
description and evaluation of the observations.

     (3)  Test report. In addition to the reporting recommendations as
specified under 40 CFR part 792, subpart J, the following specific informa-
tion should be reported:

     (i)  Species and strain used.

     (ii) Rationale for selection of species (if species is other than the spe-
cies preferred or required by OPP's toxicology  data requirements for pes-
ticide registration).

     (iii) Rationale for selections of strain.

     (iv) Source of supply of animals.

     (v) Description of any pre-test conditioning including diet and quar-

     (vi) Numbers of animals of each sex in test and control group.

     (vii) Age and condition of animals at beginning of study.

     (viii) Description  of caging  conditions  including number  (and any
change  in number) of animals per cage, bedding  material, ambient tem-
perature and humidity,  photoperiod,  and identification of diet of the test

     (ix) Description of treatments  used to prevent or control infectious
diseases if such treatment was taken during a test  or shortly before a test
was begun.

     (x) The report should indicate all ways  in which the test procedure
fails to meet  applicable  standards for acceptable testing contained in OPP's
toxicology data requirements for pesticide registration and should state the
reasons  for such deviations (see 40 CFR part 158).

     (4) Format, (i)  The test  report should include all information nec-
essary to provide  a complete and  accurate description and evaluation of
the test  procedures and results. A test report  should contain at least three
parts: A summary and  evaluation of the test results, a description of the
test procedures, and the data and information required by each applicable
section  of  OPP's toxicology data requirements for pesticide registration.

Particular information, data, or analysis may be required more than once
in the test report, and it should be referenced each time that it is required.

     (ii) Test chemical. The test report should include:

     (A)  Physical nature  and,  where  appropriate,  concentration and  pH
value for the test substance.

     (B) Chemical name, molecular structure,  and a qualitative and quan-
titative determination of its chemical  composition (including names and
quantities of known contaminants and impurities, so far as technically fea-
sible; the determinations should also include quantities of unknown mate-
rials, if any, so that  100 percent of the  sample tested is accounted for.

     (C) Manufacturer and lot number of the test substance.

     (D) Identification and composition of any vehicles  (e.g. diluents, sus-
pending agents, and emulsifiers)  or other materials used in administering
the test substance.

     (iii) Test procedures. The test report should include:

     (A)  Specification of test methods, including a full description of the
experimental design and procedures, the length of the study, and the dates
on which the study began and ended.

     (B) All dose levels administered,  method  and frequency of adminis-
tration (including hour of dosing  in relation to  photoperiod), total volume
of material (i.e., test substance plus vehicle), individual dosings, and dura-
tion  of treatment.

     (C) The method  of randomization used in  selecting samples to assay,
the assay method used  to determine the  stability and homogeneity of the
test substance being  administered, and results of the assay if the test sub-
stance is administered by a vehicle.

     (D) Include references to statistical and any other methods employed
for analyzing the  raw data, and references to any published literature used
in developing the test protocol, performing  the testing,  making and inter-
preting the observations, and compiling and evaluating the results.

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

     (1) Bermes, E.W. et al.  Chapter 2. Statistics, normal value and quality
control. Fundamentals of Clinical Chemistry. Tietz, N., ed. W.B. Saunders,
Philadelphia (1976).

     (2) Dharan,  M.  Total Quality Control in the Chemical Laboratory.
C.V. Mosby: St. Louis (1977).

     (3) Dixon, W.J. ed. Biomedical Computer Programs (BMD). 2nd edi-
tion, University of California Press: Los Angeles (1970).

     (4) Draize, J.H. Dermal Toxicity. Appraisal of the Safety of Chemicals
in Foods,  Drugs and Cosmetics.  Association of Food and Drug Officials
of the United States (1959, 3rd printing 1975) pp. 46-59.

     (5) Draize, J.H. et  al. Methods for the Study of Irritation and Toxicity
of Substances Applied Topically to the Skin  and Mucous Membranes,
Journal of Pharmacology  and Experimental Therapeutics.  83:377-390

     (6) Feigenbaum, A.V.  Total Quality Control Engineering and Man-
agement. McGraw-Hill, New York (1961).

     (7) Galen, R.S. and S.R. Gambino. Beyond Normality (The Predictive
Value and Efficiency of Medical Diagnosis). Wiley, New York (1975).

     (8) Inhorn, S.L., ed., Quality Assurance Practices for Health Labora-
tories.  American  Public Health Association: Washington, D.C.  20036

     (9) Marzulli, F.N. and Maibach,  H.I. Dermatotoxicology and Phar-
macology, Advances in Modern  Toxicology. Vol.  4.  (New  York:  Hemi-
sphere Publishing  Corp., 1977).

     (10) National Academy of Sciences.  Principles  and Procedures for
Evaluating the Toxicity of Household Substances. A report prepared by
the Committee for the Revision of NAS Publication 1138, Under the  aus-
pices of the Committee on Toxicology,  National Research  Council,  Na-
tional Academy of Sciences, Washington, DC, (1978).

     (11)  Nie,  N.H. et al.  Statistical Package  for  the  Social  Sciences
(SPSS). 2nd Ed. McGraw-Hill, New York (1975).

     (12) Oranization for Economic Coperation and Development.  OECD
Guideline 402, Paris (1987).

     (13) Reed, A.H. and R.J. Henry. Chapter 12. Accuracy, precision and
control  charts. In Clinical  Chemistry:  Principles and Technics. 2nd Ed.
Henry, R.J., ed. Harper  and Row, New York (1974).

     (14) Remington, R.D.  and M.A. Schork. Statistics with Applications
to the Biological and Health Sciences.  Prentice-Hall, New York. (Includes
a chapter  on non-parametric methods  (Chapter 12. Distribution  Free and
Nonparametric Methods) which are useful for non-normally distributed
data.) (1970)

     (15) Rohlf, F.J. and R.R. Sokal. Statistical Tables. W.H. Freeman and
Company: San Francisco (1969).

     (16)  Sokal, R.R.  and F.J. Rohlf. Biometry. Freeman,  San Francisco

     (17) Von Fraunhofer, J.A. and J.J. Murray. Statistics in Medical, Den-
tal and Biological Studies. Tri-Med Books, London (1976).

     (18) World Health Organization. Part I. Environmental  Health Criteria
6, Principles and Methods for Evaluating the Toxicity of Chemicals. Gene-
va, World Health Organization (1978).

     (19)  Young, J.R.  et al.  Classification as corrosive or  irritant to skin
of preparations containing acidic or alkaline  substances without testing on
animals. Toxicology In Vitro, 2,19 (1988).