United States        Prevention, Pesticides      EPA712-C-98-195
           Environmental Protection     and Toxic Substances      August 1998
           Agency          (7101)
&EPA    Health Effects Test
           OPPTS 870.2400
           Acute Eye Irritation

     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.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.2400  Acute eye 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 OPPTS  798.4500 Primary Eye Irrita-
tion; OPP 81-4 Acute Eye Irritation—Rabbit (Pesticide Assessment Guide-
lines, Subdivision F—Hazard Evaluation;  Human and Domestic Animals)
EPA report 540/09-82-025, 1982; and OECD 405 Acute Eye Irritation/

     (b) Purpose. (1)  In the assessment and evaluation of the toxic charac-
teristics of a substance, determination  of  the irritant and/or corrosive ef-
fects on eyes of mammals is an important  initial step.  Information derived
from this test serves  to indicate the existence  of possible hazards likely
to arise from exposure of the  eyes and associated mucous membranes to
the test substance.

     (2) Data on primary  eye  irritation are  required by 40 CFR  158.340
to support the registration of each  manufacturing-use product and end-use
product. (See § 158.50 to determine whether these data must be submitted
and which purity/grade of the test substance should be tested.)

     (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.

    Eye corrosion is the  production of irreversible tissue damage in the
eye following application  of a test substance  to the anterior  surface of
the eye.

    Eye irritation is  the production of reversible changes in the eye fol-
lowing the application of a test substance to the anterior surface of the

     (d) Principle of  the  test  method. The substance to be tested is ap-
plied in a single dose to one of the eyes  in each of several experimental
animals; the untreated eye is used  to provide control information. The de-
gree of irritation/corrosion is evaluated and scored at specified intervals
and is fully described to provide a complete evaluation of the effects. The
duration of the  study should be sufficient to permit  a  full evaluation of
the reversibility or irreversibility of the effects observed. The period of
observation should be at least 72 h, but need not exceed 21 days. Animals
showing severe  and enduring  signs of distress and pain may need to be
killed in a humane fashion.

     (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 owing to their predictable corrosive properties. Buffer capac-
ity should also be taken into account.

     (2) Materials which have demonstrated definite corrosion or severe
irritation in a dermal  study need not  be further tested for eye irritation.
It  may be presumed that  such  substances will produce  similarly severe
effects in the eyes.

     (3) Results from well validated  and accepted in  vitro test  systems
may serve to identify corrosives  or irritants such that the test material need
not be tested in vivo.

     (f) Test procedures—(1) Animal selection—(i) Species and strain.
A  variety of experimental animals  has been used, but it is recommended
that testing  should be performed using healthy adult albino rabbits. Com-
monly used laboratory strains should be  used. If another mammalian spe-
cies is used, the tester should provide  justification/reasoning for its selec-

     (ii) Number of animals. A single  animal should be considered if
marked effects are anticipated.  If  the results of this  test in one animal
suggest the test substance to be a severe irritant (reversible effect) or corro-
sive (irreversible  effect) to the eye using the procedure described, further
tests may not need to be performed. In  cases other than a single animal
test, at least three animals should  be  used. Occasionally, further testing
in  additional animals may be appropriate to clarify equivocal responses.

     (2) Dose level. For testing liquids, a dose of 0.1 mL is recommended.
In testing solids, pastes, and particulate substances, the amount used should
have  a volume of 0.1  mL, or a weight of not more  than 100 mg (the
weight must always  be recorded). If the  test material is solid or granular,
it  should be ground  to a fine dust. The  volume of particulates  should be
measured  after gently compacting  them (e.g. by tapping the measuring
container). To test a substance contained in a pressurized aerosol container,
the eye should be held open and the test substance administered in a single
burst of about 1  sec from a distance of 10 cm  directly in front of the
eye. The  dose may  be  estimated by  weighing  the container before  and
after use. Care should be taken not to damage the eye. Pump sprays should
not be used  but instead the liquid should be expelled and 0.1 mL collected
and instilled into the eye as described  for liquids. For volatile substances,
the dose may be  estimated  by  weighing the container before and after

     (3) Examination of eyes prior  to  test. Both eyes of each  experi-
mental animal provisionally selected for testing should be examined within
24 h  before testing  starts  by the same  procedure to be used during  the

test examination. Animals showing eye irritation, ocular defects, or pre-
existing corneal injury should not be used.

     (4) Application of the  test substance, (i) The test  substance should
be placed in the  conjunctival sac of one eye of each animal after gently
pulling the lower lid away from the eyeball. The lids are then gently held
together for about 1 sec in order to limit loss of the material. The  other
eye, which remains untreated, serves as a control. If it is thought that the
substance may cause extreme pain, local anesthetic may be used prior to
instillation of the test substance. The type and concentration of the local
anesthetic should be carefully selected to  ensure that no significant dif-
ferences in reaction to the test substance will result from its use. The con-
trol eye should be similarly anesthetized.

     (ii)  The  eyes  of  the test animals  should not be washed out for
24 h following instillation of the test substance. At 24 h, a washout may
be used if considered  appropriate.  This is to show whether  washing with
water palliates or exacerbates irritation.

     (iii) For some substances shown to be irritating by this test, additional
testing using animals with eyes washed soon after instillation of the sub-
stance may  be indicated.  Half a minute after instillation, the  eyes of the
animals are washed with water for 30 sec, using a volume and velocity
of flow which will not cause  injury.

     (5) Observation  period.  The  duration of the observation period is
at least 72  h,  and  should not be  fixed rigidly, but should be  sufficient
to evaluate fully the reversibility or irreversibility of the  effects observed.
The observation period normally need not exceed 21 days after instillation.

     (6) Clinical  examination and scoring, (i) The eyes should be exam-
ined at 1, 24, 48,  and 72 h. If there is  no evidence of irritation  at 72
h, the study may be ended. Extended observation (e.g. at 7 and 21  days)
may be necessary if there is persistent corneal involvement or other ocular
irritation in  order to determine the progress of the lesions and their revers-
ibility or irreversibility. In addition to the observations of the  cornea, iris
and conjunctivae, any other  lesions which are  noted should be  recorded
and  reported. The  grades of ocular  reaction using the  following  table
should be recorded at each examination.

                       Grades for Ocular Lesions
  Opacity: Degree of density (area most dense taken for reading). No ulceration
    or opacity                                                                     0
  Scattered or diffuse areas of opacity (other than slight dulling of normal luster),
    details of iris clearly visible                                                      *1
  Easily discernible translucent area, details of iris slightly obscured                      *2
  Nacrous area, no details or iris visible, size of pupil barely discernible                   *3
  Opaque cornea,  iris not discernible through the opacity                               *4
  Normal                                                                         0
  Markedly deepened rugae, congestion, swelling  moderate circumcorneal  hy-
    peremia, or injection,  any of these or combination of any thereof, iris still re-
    acting to light (sluggish reaction is positive)                                        *1
  No reaction to light, hemorrhage, gross destruction (any or all of these)                 *2
  Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and
  Blood vessels normal                                                             0
  Some  blood vessels definitely hyperemic  (injected)                                    1
  Diffuse, crimson  color, individual vessels not easily discernible                         *2
  Diffuse beefy red                                                               *3
  Chemosis (refers to lids and/or nictitating membranes)
  No swelling                                                                     0
  Any swelling above normal (includes nictitating  membranes)                            1
  Obvious swelling with partial eversion of lids                                         *2
  Swelling with lids about half closed                                                 *3
  Swelling with lids more than half-closed                                             *4

  *Starred figures indicate positive grades.
            (ii)  Examination of reactions can be facilitated by use of a binocular
       loupe, hand slit-lamp, biomicroscope,  or  other suitable device. After re-
       cording the  observations at 24 h, the  eyes of any or all  rabbits may be
       further examined with the aid of fluorescein.

            (iii) The grading of ocular responses is subject to various interpreta-
       tions. To promote harmonization  and  to  assist  testing laboratories  and
       those involved in making  and interpreting the observations, an illustrated
       guide in  grading eye irritation should be used.

            (g)  Data and  reporting—(1) Data summary.  Data  should be sum-
       marized  in tabular form, showing for each individual animal the irritation
       scores at observation time up until reversal (nonpositive grades) or 21 days
       when the test is concluded; a description of the degree and nature of irrita-
       tion;  the presence  of serious lesions  and any effects other than  ocular
       which were observed.

            (2)  Evaluation of the results.  The ocular irritation scores should be
       evaluated 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 and are only meaningful when  supported by a  full
       description and evaluation  of the observations.

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

     (i)Species, strain, sex, age, and source of test animal.

     (ii) Rationale for selection of species (if species is other than the spe-
cies preferred.

     (iiii) Tabulation  of irritant/corrosive  response data for each individual
animal at each observation time point (e.g. 1, 24, 48, and 72 h until revers-
ibility of lesions or termination of the test).

     (iv) Description of any lesions observed.

     (v) Narrative description of the degree and nature of irritation or cor-
rosion observed.

     (vi) Description of the method used to score  the irritation at 1, 24,
48, and 72 h (e.g. hand slit-lamp, biomicroscope, fluorescein stain).

     (vii) Description of any nonocular effects noted.

     (viii) Description of any pre-test conditioning, including diet,  quar-
antine, and treatment  of disease.

     (ix)  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 test ani-

     (x)  Manufacturer, source, purity, and lot  number of  test substance.

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

     (xii) Identification,  composition, and characteristics of any vehicles
(e.g., diluents, suspending  agents, emulsifiers, and anesthetics)  or  other
materials used in administering the test substance.

     (xiii) A list of references  cited in the body  of the report,  i.e., ref-
erences to any published literature used in developing the test protocol,
performing the testing, making and interpreting observations, and compil-
ing and evaluating the results.

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

     (1) Buehler, E.V. andNewmann,  E.A. A Comparison of Eye Irritation
in Monkeys and Rabbits. Toxicology and Applied Pharmacology 6:701-
710 (1964).

     (2) Draize, J.H. Dermal Toxicity. Appraisal of the Safety of Chemicals
in Foods,  Drugs and Cosmetics. The Association of Food and Drug Offi-
cials of the United States (1959) 3rd printing 1975, pp. 49-52.

     (3) Draize, J.H.  et al. Methods for the study of irritation and toxicity
of substances applied topically to the skin and mucous membranes. Jour-
nal of Pharmacology and Experimental Therapeutics. 83:377-390 (1944).

     (4) Loomis, T.A. Essentials of Toxicology. Lea and Febicer, Philadel-
phia 3rd ed. 1978 pp. 226-232.

     (5) Kay, J.H. and Calandra, J.C., Interpretation of eye irritation tests.
Journal of the Society of Cosmetic Chemists 13:281-289 (1962).

     (6) 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 (1977).

     (7) World Health Organization. Part I.  Environmental Health Criteria
6. Principles and Methods for Evaluating the Toxicity of Chemicals. World
Health Organization, Geneva (1978).