HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
GUIDANCE FOR EVALUATION OF EYE IRRITATION TESTING
Prepared by
Frank J. Vocci
and
Van M. Seabaugh
Standard Evaluation Procedures Project Manager
Orville E. Paynter, Ph.D., D.A.B.T.
Hazard Evaluation Division
Office of Pesticide Programs
United States Environmental protection Agency
Office Of Pesticide Programs
Washington, D.C. 20460
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the development of the test, animal models, scoring system,
labeling, and factors affecting the results. Tl ere are dis—,
cussions on objective and alternative technologies, evalatiøn
based on weight—of—evidence, epidemiological data, and low
dose and dose response studies. A tier system is presented .
incorporating present methods and possible future altern—
tives. Data Reporting Guidelines (Subdivision F, Series
81—4, Eye Irritation) are available [ Natiot3al Technical Infor-
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This Standard Evaluation Procedure (SEP) is one of a set
of guidance documents which explain the procedures used to
evaluate environmental and human health effects data submitted
to the Office of Pesticide Programs. The SEPs are designed
to ensure comprehensive and consistent treatment of major
scientific topics in these reviews and to provide interpretive
policy guidance where appropriate. The Standard Evaluation
Procedures will be used in conjunction with the appropriate
Pesticide Assessment Guidelines and other Agency Guidelines.
While the documents were developed to explain specifically
the principles of scientific evaluation within the Office of
Pesticide Programs, they may also be used by other offices in
the Agency in the evaluation of studies and scientific data.
The Standard Evaluation Procedures will also serve as valuable
internal reference documents and will inform the public and
regulated community of important considerations in the
evaluation of test data for determining chemical hazards. I
believe the SEPs will improve both the quality of science
within EPA and, in conjunction with the Pesticide Assessment
Guidelines, will lead to more effective use of both public
and private resources.
Anne L. Barton, Acting Director
Hazard Evaluation Division
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Table of Contents
Preface . .. . . . . . . . . . . . . • • • •• • • • • SI ••
Eye Irritation Studies 1
A. Ilistorical Background ..................... 1
B. AnatomicalConsiderations......... 1
1. Conjuncti.va .. •.. 1
2. Cornea . • • 1
3. Iris 3
C . E x p e r i men t a 1 Mode 1 S . . . . . . . . . . . 4
1. Albino Rabbit •. . . . . 4
2. Other Species Used For Testing 4
3. EvaluationOfOcularTesting.......... 5
a. ScoririgSystems 5
b. Factors Affecting Results 5
D. CoinpietionOf Evaluation 6
1. Objective And Alternative Technologies 6
2. Evaluation Based On The
Weight—Of—Evidence 9
3. EpidemiologicalData......................... 10
4. Low Dose Testing And Dose Response
Studies .. . . 11
E. Toxic Classification Of Compounds Based On The
Weight—Of-Evidence 12
F. Tiersystem—FutureTestirig..................... 12
i C . .
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Page
Table 1: Corneal Thickness Of Several Species
of Animals . . . . . . . . . . . . . . . . . 13
Table 2: Comparison Of Response Of The Rabbit
and Monkey Eye to Irritants ............ 14
Table 3: Eye Scoring Scheme Based On The Use
Of A Slit Lamp Biomicroscope and
Fl orescein . . ..... . . . . . •.. . . . . . . . . 15
Table 4: Label Statements Regarding Eye
Irritation Haiards Due To
Pesticid s . •.... 16
Fi.gurel: EyeAnatomy................... 19
Figure 2: Proposed TestLng Scheme ............... 20
Definitions For Eye Irritation Testing ..... . 21
Bib1 ography . .. . . . •IISI . 23
Li
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Preface
Art application for registration of a pesticide requires
toxicity data from which short and long term risks may be
ascertained. Within the body of short term studies, the
potential for a pesticide to produce eye irritation is an
uppermost consideration in the assessment process. These
concerns culminated in November 1982 when the EPA’S Office
of Pesticide Programs Issued testing guidelines (Subdivision F:
Pesticide Assessment Guidelines, Hazard Evaluation — Human and
Domestic Animals) and Series 81—4 of these guidelines provides
the basis for eye irritation testing.
Data on eye irritation are required by the Code Of Federal
Regulations (40 CFR part 158) to support the registration of each
manufacturing—use product and each end-use product. The “Pesti-
cide Assessment Guidelines” (EPA, 1982) further discusses these
requirements. Refe’r specifically to 40 CFR 158.50 and 158.135
to determine if these data must be submitted. The section of
40 CFR 158.50 entitled “Formulators Exemption” requires a
registrant of a manufacturing-use product to submit (or cite) any
data pertaining to the safety of an active ingredient if the same
data are required to support the registration of an end—use
product that could legally be produced from the registrant’s
manufacturing—use product.
The purpose of this Standard Evaluation Procedure (SEP) is
to provide supplementary information to S81—4. This document
further assumes that human experience or epidemiological evidence
is wanting, and judgments of classifications are wholly dependent
upon animal tests. Therefore, the basis for classification may
be superseded when valid, epidemiological evidence outweighs the
surrogate test which previously had demonstrated either false
negative or false positive results. Without the epidemiological
evidence, it is anticipated that sufficient guidance for classi-
fications is provided with this test.
iii
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Eye Irritation Studies
A. Historical Background
Ophthalmic toxicity reviews are available (EPA, 1981;
McDonald et al., 1983; NAS, 1975). Mann and Pullinger (1942)
described the use of rabbits to predict ocular toxicity of test
substances in humans. These latter authors advocated the use
of pigmented eyes rather than non—pigmented ones (albino rabbits),
and relied on description of individual animal responses to
address the irritant properties of test substances. Friedenwald
et al. (1944) reported an albino rabbit method of assessing ocular
toxicity that provided a scoring system based on the description
of individual animal responses. Draize et al. (1944, 1955, 1959)
modified Friedenwald’s procedure, and published an eye irritancy
grading system to further assist the evaluation of ocular toxicity
of test substances. With the passage of the Federal Hazardous
Substances Act (FHSA) in 1958, a modified “Draize” procedure
became the required test and illustrated eye guides have been
published as further aids in training (FDA 1965; CPSC, 1976;
EPA, 1981).
B. Anatomical Considerations (Bloom and Fawcett, 1975);
( Figure 1 )
1. Conjunctiva
The conjunctiva is the nonkeratinized squamous
epithelium containing mucous secreting cells that covers the
anterior sciera (bulbar conjunctiva) and inner surface of the
eyelids (palpebral conjunctiva). It is continuous with the
epidermis at the lids and the cornea at the limbus. The palpebra].
conjunctiva is more stratified and contains more goblet cells
than the bulbar conjunctiva. Glycoproteins from the conjunctiva
contribute to the stability of the tear film (Holly and Lemp,
1971).
2. Cornea
The cornea is an avascular transparent tissue
which is inserted to the sciera at the limbus. From anterior
to posterior the cornea has five distinct cell layers; the
epithelium, Bowman’s membrane, stroma, Descemet’s membrane, and
the endothelium. The epithelium, a nonkeratining stratified
squamous type, is composed of five to six layers. The basal
layer is attached to the basement membrane by numerous hem—
idesomosomes and is composed of cuboidal cells which are
closely packed. The cells of the middle layers are polygonal
in shape and bordered by many interdigitations attached by
desmosomes. The most superficial layer of cells is squamoid
in shape and covered with numerous microvilli (Phister, 1973).
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Bowman’s membrane, a modified portion of the anterior
stroma, is a clear acellular layer whose thickness varies among
species. This structure at a thickness of 2 urn in rabbits is
essentially unrecognizable but is prominent in humans arid
other primates. The thickness of this structure is humans
has been reported as 8 to 12 urn (Prince et al., 1960).
The stroma accounts for 90 percent of the thickness of
the cornea. It is composed of parallel laniellae of collagen
I ibrils which run from limba]. border to limbal border. Numerous
keratocytes are scattered throughout the collagen fibrils. The
diameter of collagen fibrils increases at the limbus where the
fibers insert into the scleral stroma and begin to assume an
interdigitated and twisted appearance.
The next layer, Descemet’s membrane, is a clear elastic
membrane of the finpl layer of the cornea, the endothelium.
Descemet’s membrane shows little interspecies variation in
thickness of structure.
The endotheliuin is composed of a single layer of
squamous cells the apical surfaces are tightly attached by
Zonu].a Acciudentes. The cells form a hexagonal array when viewed
via slit lamp, and are 4 to 5 urn thick in humans and slightly
thinner in other species. Their function is extremely important
in maintaining the relative state of dehydration of the cornea].
stroma. For this reason, damage to the endotheliuni is more
serious than damage to the epithelium.
Comparatively, the corneas of animals differ from those
of humans in several ways. The rabbit cornea is thinner than
the human cornea with most of the difference accounted for by
the thickness of the stroma (Table 1). The collagen fibrils are
more loosely packed in rabbits than in humans. As previously
stated, Bowman’s membrane is thinner in other animals than humans.
Examination of the cornea for irritant effects includes
evaluation of both stromal lesions and epithelial lesions.
Large epithelia] - defects are readily identifiable by slit lamp
biomicroscopy whereas smaller defects are easily visualized by
the addition of fluorescein sodium drops and the use of a cobalt
blue filter on the slit lamp. Epithelial lesions may occur
independently of stromal lesions provided the basal cell layer
remains intact. Stromal lesions are detected as opacities which
r%preSer%t edema/infiltration of t te comas by inflammatory eells.
Increasingly severe stremal. lesions are indicated by an increase
in opacity area of corneal involvement.
Prolonged corneal edema induces proliferation of
endothelial cells at the limbus resulting in the formation
of new vessels extending toward the center of the cornea.
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Neovascularization of the cornea (pannus), occurs readily in
rabbits and represents a permanent change secondary to ocular
insult by irritants. Permanent corneal opacities may also
result from severe epithelial and stromal lesions.
The penetration of chemical compounds into the cornea
is biphasic. Lipid soluble substances can readily pass through
the corneal epithelium and hydrophilic substances can pass through
the stroma. A test compound must have a lipid soluble and water
soluble phase to penetrate the cornea effectively.
Studies by Cogan and Hirsch (1944) and Cogan et al.
(1944) have shown that the absence or presence of charge alone
could not account for differences in permeability and that
molecular size of a test compound correlated only approximately
with corneal penetration. They further concluded that the
corneal penetration of weak electrolytes was facilitated when
the dissociation co’nstant of the compound is small so that the
test compound is present in an undissociated state at the pH
of the eye and when the undissolved form of the test substance
possesses lipophilic characteristics.
Krueger (1959) verified the findings of Friedenwald
et al. (1944, 1946) that the corneal epithe].ium represents a
barrier to the penetration of acids. Alkalies penetrated into
the anterior chamber more quickly than acids in isolated pig
eyes with intact corneal epithelium, but the rates of penetra-
tion of acids and alkalies were approximately the same when the
corneal epithelium was removed. For this reason, the early
assessment of ocular damage by acid burns is a measure of long—
term damage to be expected (Potts and Gonasun 1975) whereas
alkali burns result in progressive lesions so that initially
‘mild lesions frequently develop into severe lesions as a late
complication (Hughes 1946).
3. Iris
The iris is the anterior extension of the ciliary
body. Thusly, ocular irritants applied topically to the cornea
may produce pathologic changes in the iris.
Although not required by EPA, the slit lamp may be
used to further ascertain eye injury (Baldwin et al., 1973).
Injury to the iris in irritation studies is monitored by observing
injection in the vessels, the thickness of the iris stroma, and
aqueous flare. The vessels of the iris become hyperemic following
irritation. Leakage of fluid from the unfenestrated capillary
endothelium in the stroma results in edema recognizable in the
slit lamp as a swelling of the iris. The Tyndall phenomenon,
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commonly called aqueous flare, results from the release of
proteinaceous material or cells into the aqueous humor. In
the normal aqueous chamber, the light beam is not discernible.
However, the proteiriaceous material released during iritis
changes the refractive index of light so that the light beam
from a slit lamp becomes visible as it passes through the aqueous
humor. Aqueous flare is, therefore, presumptive evidence of a
breakdown in the blood aqueous barrier. The order of changes in
iris irritation in increasing severity is hypereniia of vessels,
stromal edema, and aqueous flare. The presence of inflammatory
cells and aqueous flare are diagnostic of frank iritis.
C. Experimental Models
1. Albino Eabbit
An important criterion in selecting an animal or
test model to evaluate potentially hazardous compounds is the
ability of the animal test to predict the human response. Ideally,
this animal would exhibit similar sensitivities to a wide range
of chemical compounds, and would be docile, inexpensive, and
readily available. Although several species of animals including
dogs and rhesus monkeys have been used in ocular testing, the
albino rabbit has several advantages and has been used most
frequently. The albino rabbit is easy to handle and maintain and
is relatively inexpensive. The corneal surface and conjunctiva
are large and easily seen. The unpigmented iris is easy to
evaluate in terms of congestion of iris vessels. A large data
base exists for rabbits.
A review of the literature of ocular testing in
albino rabbits reveals that this animal exhibits similar or more
sensitive responses for many test compounds, less sensitive
responses for a few test compounds, and totally failed to predict
an irritant response for several compounds (Carter, 1906; Lewin
and Guillery, 1913; Leopold, 1945; Carpenter and Smyth, i946
Hogiwara and Sugiwia, 1953; Grant, 1974; van Abbe, 1973; Marsh
and Maurice, 1971).
In contrast to the above reports where the albino
rabbit accurately predicted the human response, there are several
reports where the sensitivity of the albino rabbit to test
compounds was either greatly diminished or nonexistent (Estable,
1948; Gartner, 1944; Lewin and Guillery, 1913; Marsh and Maurice,
l97 .; Grant, 1974W Van Abbe, 1973 Beckley et al. 1969).
2. Other Species Used for Testing
The anatomical differences between the human and
rabbit eye and the failure of the rabbit to predict the human
response in several instances of ocular testing have led to
criticism of the use of rabbits as the experimental model in
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ocular toxicity testing. The use of primates has been suggested
on the basis of anatomic similarities. However, it must be
remembered that primates are expensive, difficult to obtain,
costly to maintain, and not always easy to restrain. In general,
studies of the comparison of responses of rabbits and primates
to ocular irritants have shown that primates more closely predict
the human response, and the rabbit is usually the most sensitive
species (Beuhier and Newmann, 1964 Beckley, 1965; Beckley et a ] ..,
1969; Green et a].., 1978; Hirst et a].., 1981).
From the literature cited, it is concluded that the
rabbit is a more sensitive species, but important exceptions
exist to the general rule (Table 2). For this reason, one may
consider the albino rabbit to be a very good, but not a failsafe
test model. Data derived from rabbit tests may not be directly
extrapolated to man in terms of anatomical location and/or
severity of irrita!7cy. The use of other species for testing
purposes may be justified in some instances. Furthermore,
epidemiological data as to potential exposure in humans must
be considered (e.g., compounds dispersed as aerosols in the
environment should be tested as aerosols).
3. Evaluation of Ocular Testing
a. Scoring Systems
The grades obtained from ocular testing are
used for labeling purposes (CPSC 1976, EPA 1982).
b. Factors Affecting Results
Marzulli and Ruggles (1973) conducted a
collaborative study using 10 laboratories. Each laboratory
tested seven materials instilled directly on to the cornea and
rated the ocular responses in terms of cornea]. opacity, chemosis,
conjunctival injection, and iritis at 1, 2, 3, and 7 days. All
laboratories were capable of distinguishing irritants when all
four criteria were employed but varied widely in separating
irritants from nortirritants when a single criterion was used.
McDonald and Shadduck (1977) reported the results
of a statistically based experimental study designed to determine
variability among a group of investigators and within a single
investigator. Analyst uniformity among three investigators was
considered very good for all ocular parameters except congestion
and discharge (e.g., correlation coefficients for cornea]. opacity
and cornea]. area tanged from 0.90 to 0.97). Analyst precision
for each of three investigators ranged from 0.73 to 0.88. The
authors reported that the scores obtained were acceptable and
stressed that reliability was achieved by trained investigators.
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Seabaugh et aL (1976) evaluated the length of
exposure and the degree of chemically induced eye irritation
in albino, New Zealand Rabbits. This study evaluated the utility
of ocular irrigation to determine if such a procedure improves
the predictive capability of the rabbit eye irritation test. All
of the 32 chemicals tested were compared for effects of length of
exposure and a 2 minute irrigation. In comparing 30 second vs.
24 hour exposure groups for irritancy changes, 20 percent of the
chemicals decreased, 5 percent increased, and 75 percent showed
no changes. With 2 minute vs. 24 hour exposures, 15 percent
decreased, 10 percent increased, and 75 percent had no change.
For 5 minute vs. 24 hour exposures, 13 percent decreased, 10
percent increased, and 77 percent did not change. For 6 chemicals
tested, there were predominantly no apparent benefits from washing
rabbit eyes with either 2 or 5 minute irritation at any of the
chemical exposure times as compared to 24 hour exposures. With
the exception of 3 powders among all of the other irritant chemicals
tested, rabbit ocular responses did not change to the category of
non—irritant following shorter exposures. This data indicated
that irrigation of rabbit eyes does not contribute to the outcome
of irritant/nonirritant eye classification categories for 16 CFR
1500.42 (Federal Hazardous Substance Act Eye Tests).
An additional factor affecting subjective scoring
is the manner in which data are reported. Ballantyne and Swanston
(1972) maintained that summed ocular scores are uninformative
about individual tissue responses because identical mean scores
could be obtained for two entirely different reactions. Shuster
and Kaufman (1974) stated that the addition of scores assigned to
different tissues is statistically improper because some lesions
are more important than others (e.g., mild but persistent cornea]
lesions are more threatening than severe transient conjunctival
injection. Ocular scores must be reported separately for each
ocular tissue, as indicated in the illustrated guide (FDA, 1965).
D. Completion of Evaluation
1. jective and Alternative Technologies
The previously described subjective tests have long
been criticized for a lack of standardization in methods and
assessment. The lack of uniformity in testing methods has
provided the impetus to develop objective methods of in vitro
assessment of ocular toxicity. One such method is the method
of corneal thickness by use of a pachorneter (Mishima and Redbys
1968). Although the technique adds to the time and expense
of ocular testing, it has the advantages of being both highly
reproducible and noninvasive (Burton, 1972: Conquet et a].,
1977).
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Another line of investigation in the objective
measurement of ocular toxicity is the measurement of intraocular
pressure (10?). Increases in 10? have been reported by several
investigators following application of known eye irritants (Walton
and Heywood, 1978; Ballantyne et al., 1972; Maul and Sears, 1976).
Histopathological analysis, while not truly an
objective technique, is a sensitive and reliable technique for
evaluation of ocular pathology (Weitman et al., 1965; Green
et a].., 1978).
Several additional techniques have been successfully
employed to evaluate objective changes following the application
of known irritants to the rabbit eye. Maul and Sears (1976)
measured pupil diameter and the concentration of protein in
the aqueous humor of rabbit eyes following exposure to nitrogen
mustard. Jampol et al. (1975) had previously shown that the
response to nitrogen mustard depends on sensory innervation and
is not mediated by prostaglandins to any significant degree.
Maurice (1968) described a modification of slit—lamp
biomicroscopy that he termed specular microscopy. The addition
of this apparatus allows one to view and photograph endothe].ial
cells of the cornea. Sugar (1979) has reviewed the use of the
specular microscope in ophthalmological investigations. The
utility of this instrument is in providing information and a
photographic record of the effects of potential eye irritants
on the corneal endothelaum.
The use of scanning and transmission electron
microscopy may prove useful in evaluating toxic effects of
potential irritants and topical ocular products (McDonald and
Shadduck 1977). However, Bursteiri (1980) pointed out that the
major drawback of electron microscopy is the requirement of
chemically fixed tissue in the dehydrated state. Electron
microscopy is also expensive, time consuming, subject to sampling
error, and requires highly skilled personnel. It probably finds
its best application in the investigation of the process of
ocular irritation and not the determination of irritancy for a
specific test compound.
Both the subjective and objective testing parameters
currently employed in ocular irritation rely on the use of intact
animals for the endpoint determinations. There has been a large
investigative effort to move toward the use of cell and organ
cultures to determine the irritant potential of test compounds.
The aim is to develop in vitro testing methods that would greatly
reduce or elimInate entirely the need for anImal testing. Such
bioassay methods would provide objective data that is rapidly
obtained, cost effective, and subject to standardization. This
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line of investigation has diverged along two tracks, biochemical
assays and morphologic assays. The biochemical assays will be
summarized first, and followed by a summary of the morphologic
methods.
Gasset et al. (1974) employed enzyme histochemistry
to determine the effect of ophthalmic preservatives on rabbit
eyes. Staining of NADH-2 oxidoreductase was employed to deter-
mine the viability of the corneal endothelium.
Borenfreund et a].. (1983) described an in vitro
assay based on the reduction of 3H—uridine uptake in HEP-G2,
an established human hepatoma cell, and Balb/c 3T3, a mouse
fibroblast cell line. A similar method, detailing a technique
for determining irritant potential by ATP measurements was
described by Kemp et al. (1985). Sciafe (1985) described a
f] .uorometric and membrane bound enzyme released assay for
determining in vitro the cytotoxic potential of surfactants.
Other bioche Tcal assays were investigated (Goldberg 1985).
The University of Washington studied the release of plasminogen
activator by corneal cells after exposure to irritants. The
preliminary data suggested that corneal. cell cultures release
plasminogen activator in response to exposure to eye irritants.
A similar line of investigation was pursued at johns Hopkins
University. Using Chinese hamster ovary cells, the potential
of irritants to release hydrolytic enzymes from the fibroblasts
was studied (Goldberg 1985).
Since irritants produce nonspecific cytotoxic
effects, the damage produced may be simulated by in vitro studies
which use a variety of endpoints. These endpoints for toxicity
include cell viability as determined by trypan blue and fluorescence
(Kemp 1985), morphological changes (Shopsis 1985), cell detachment,
cloning efficiency (Reinhardt et al. 1985), and cell membrane
integrity (Sciafe 1985).
Other morphological techniques applied to the problem
of in vitro detection of irritants would include the following:
Cytologic and colony inhibition assays employing Balb/c 3T3 cells.
Semiconfluent cell cultures are exposed to the potential irritant
for 24 hours then examined by phase microscopy for evidence of
toxicity such as vacuolization and blebs. The results of these
assays have correlated well with data obtained from the Draize
test (Shopsis et al. 1985). Reinhardt et al. (1985) reported on
a comparative study of 3 different cell culture lines exposed to
57 potential irritants. Their endpoints were cell detachment as
determined by an automated cytometer, cloning efficiency, and
growth inhibition. The results showed that all three cell lines
and three endpoints were equally effective in determining the
rank order of irritants. Muir et al. (1983) investigated the
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hemolytic potency and ability to block spontaneous contractions
in isolated mouse and rabbit ileum of eight surfactants. The
results showed that hemolytic potency failed to correlate with
in vivo ocular testing and the isolated rabbit ileum preparation
gave the best correlation with in vivo findings in the rabbit
eyes. Additional morphologic—based tests are in various stages
of development (Goldberg 1985). Human cornea ] . cell lines were
evaluated at the Eye Research Institute in Boston for their
ability to predict irritant potential. Cytotoxicity is evaluated
by phase microscopy, trypan blue exclusion, and long—term survival
as determined by repeated cell counts.
Although not strictly an in vitro test, the chick
chorioallantoic membrane (CAM) has been proposed as a model for
irritancy testing (Leighton et al. 1985: Luepke, 1985). However,
Parish (1985) maintains that several problems exist in the CAM
in the ability to redict for pure chemicals. Known irritants
did not produce a dose response whereas several nonirritants
killed most embryos and produced necrosis in the CAM test.
The problem of validity of in vitro tests as screening
tools for eye irritancy is not confined to the CAM test, but is a
problem for all. in vitro tests at present. The ability of these
tests to be used as large—scale screening mechanisms that would
reduce the need for animal testing awaits the conformation that
will come only from large scale testing and refinement in the
techniques. The problem of validity was addressed by the FRAME
(Fund for Replacement of Animals in Medical Experiments) laboratory
in Nottingham, England (Balls and Homer 1985). This laboratory
devised a scheme for validation of in vitro alternative methods
that includes a set of coded chemiclls to be used in blind trials
of in vitro tests while summaries of the in vivo toxicity of the
test compounds are being produced. \pata submitted from blind
testing will be evaluated for interlaboratory variation and its
correspondence to in vivo testing data by an independent assessment
team. Bosshard (1985) stated that only a small number of compounds
have been tested to date by the newly developed in vitro methods;
for this reason, there is a current drought of information on the
interlaboratory variation in testing and the significance of the
test data for predicting untoward effects in man. The systematic
comparison of in vitro and in vivo data will be required to answer
the issue of validity. Epidemiologica]. data gained from accidental
exposure to chemicals will be required for fine tuning of the
estimation of the human response to irritants derived from
in vitro data. While it is doubtful that a single in vitro
test will fulfill the requirement for all test compounds, it
is entirely possible that a battery of in vitro tests as proposed
by Shopsis et al. (1985) will reduce anii al testing to a minimum.
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2. Evaluation Based on the Weight of Evidence
Toxicity in ocular tissues is synonymous with
corrosive/irritant effects. These effects are qualitative, and
not subject to the decision making process on the basis of numbers.
It is for this reason that one must use their professional judgment
to accept or reject data and to articulate the rationale for
doing so. When data are ambiguous and nonconclusive, that is,
one of six animals shows a positive response of significant
duration, the reviewer should base his/her decision on the nature
and duration of the response rather than the number of animals
responding. The reviewer must then provide the toxicity category
of the pesticide which can be interpreted from Table 4. In cases
where both corneal involvement and irritation are observed, the
test material should be placed in the highest category based on
the duration of the response. The labeling of that test material
is clearly then based on both the effects produced and their
duration, and not on the number of animals responding. For this
reason the EPA has adopted the NAS recommendation that the
observation period in eye testing be extended to 21 days (NAS
1977; Campt, 1981). considering the dose of 100 uL per eye,
data which are not indicative of an all-or—none response are
suspect, and one must. carefully evaluate the effects in the
positively responding animals for proper classification.
For a valid eye irritation test, at least 6 rabbits
must survive the test for each substance. A trial test on three
rabbits is suggested. No further testing is necessary if the
test substance produces corrosion, severe irritation, or no
irritation. If the data for a test compound shows only a mild
transient conjunctivitis (2 or 3 days) in one or more animals
with no delayed type reaction, then no further testing is required.
However, if equivocal responses occur, testing on at least 3
additional animals should be performed. If the test substance
is intended for use around the eye, then testing on at least
6 animals 5hould be performed.
3. Epidemiological Data
Data derived from small animal testing is presumably
predictive, but not necessarily indicative of the human response.
EpiderniOlOgiCal data acquired from accidental exposure should be
considered retrospectively and classification of compounds adjusted
accordingly. Chiorpromazine, a phenothiazine tranquilizer that
is still currently used clinically, was extensively tested in
both animals and humans before its approval by the FDA in 1953
and found to be noninjurious to the eye (r’iarzulli, 1968). Clin-
ical experience has shown that chlorpromazine induced deposits in
both the cornea and lens of patients. Similar experience with a
related compound, thorazine, has shown that it can produce cata-
racts and an increase in retinal pigmentation. The importance of
—10—
-------
these clinical observations underscores the fact that animal
testing is only presumed to be predictive and the true definitive
answer usually unfolds only with extensive human experience.
McLaughlin (1946) detailed 602 cases of eye burns
resulting from accidental exposure and reported that the most
serious injuries were caused by highly acidic and basic materials.
Among these patients, 7 injuries involved loss of vision whereas
458 were mild and cleared within 48 hours the remainder required
up to 10 days to heal. Clinical studies of this nature would
greatly facilitate the expansion of the data base were they to
include information on the type of compound, its concentration,
and the exposure conditions. Evidence as to the irritancy
potential of a compound derived from epidemiological studies
and clinical experience should be considered prima faciae
evidence that supersedes data derived from animal testing.
Epidemiological daba should, therefore, receive the highest
consideration in the reclassification of a test compound.
4. Low Dose Testing and Dose Response Studies
Williams (1985) conducted a study of seven known
irritants appi d directly to the cornea of albino rabbits in
volumes of 10 uL. The results showed that the severity of the
reaction was reduced, but the rank order and ability to predict
irritancy were not altered. The results were in agreement with
a prior study of seven different compounds reported by Williams
et al. (1982). A dose-response relationship was clearly established
in terms of both duration of effect and maximal score for test
compounds used at volumes of 10 ul and 100 ul. Again, test
sensitivity was not compromised and the rank of severity remained
unchanged for the 10 ul dose results. Griffith et al. (1980)
performed a dose response study of eye irritation employing 21
test compounds at 4 different doses applied to the eyes of albino
rabbits and scored for a 21—day observation period. By comparison
of the maximal score and duration of effect with the best available
data from human experiments, this group concluded that 10 ul
represents a more realistic dose for hazard testing than the
100 ul currently employed by the Draize protocol. This reduced
volume recommendation was considered at a recent meeting of the
‘OECD in April 1986. The United States, the United Iingdom, and
Switzerland supported the recommendation whereas opposition was
expressed by France, Germany, Belgium, Spain, 3apan, and The
Netherlands. The use of the low—volume test as a screening test
was discussed but not adopted as an cfl cial recommendation by
the working group (Murphy 1986). It is EPA’s (FIFRA) policy to
insist on a dosage of 0.1 mL as stated in the rabbit eye irrita-
tion testing guideline until such a time that there is sufficient
scientific data supporting the reduced volume. When this data
becomes available, a recommendation will be made to EPA to change
the guideline.
—11—
-------
F. Toxic Classification of Compounds Based on the
Weight of Evidence
Several definitions for the classification of test
compounds have been proposed. According to 40 CFR Part 158,
the difference between an eye irritant and a corrosive compound
is that the corrosive produces irreversible tissue damage to
the eye whereas the eye irritant produces changes which are
reversible. It should be realized both irritants and corrosives
produce cytotoxic effects that result in inflammation which in
the case of corrosives yield either rupture of the globe, long-
term erosions, or healing with scar formation. EPA uses a
classification of irritants and corrosives based on both effect
and duration of effect on ocular tissues (Table 4).
Beckley et al. (1969) proposed an eye irritant
classification system based on fluorescein staining and slit
lamp biomicroscopy. Although it is not part of EPA guidelines,
it is instructive in that it details guidelines for acceptance
of irritants based on minimal effects of short duration arid more
ominous effects that should alert oneself to the necessity for
labeling a compound as hazardous (Table 3).
F. Tier System — Future Testing
Current EPA guidelines mandate that compounds to be
registered be tested as manufacturing end—use products (40 CFR
Part 158). These guidelines state strongly acidic or basic com-
pounds with a pH of 2 or less or 11.5 or greater need not be
tested owing to their predictive corrosive properties. Also,
compounds that have been demonstrated as corrosives and irritants
by dermal testing need not be further tested for eye irritation.
It may be presumed that these substances could produce similarly
severe effects in the eye. Williams (1984) found that 45 of
60 dermal irritants produced severe or moderate irritation in
ocular testing. The remaining 15 skin irritants produced mild
effects which cleared within 3 days. Perhaps the greatest
change in the future testing will be the introduction of in
vitro tests which have been covered, in part, in this document.
These in vitro tests will require further research and more
extensive testing for validation and to determine their overall
utility in the risk assessment process. A prospective scheme
for future testing might be as shown in Figure 2.
—12—
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Table 1 : Corneal Thickness of Several Species of Animals.
Species Thickness (mm) References —
cat 0.62 Marzulli and Simon (1971)
Dog 0.55 Marzulli and Simon (1971)
Rhesus Monkey 0.52 Marzulli and Simon (197])
Rabbit 0.37 Marzulli and Simon (1971)
Mouse 0.10 Davson (1962)
Human 0.51 Maurice and Giardini (1951)
—13—
-------
Rabbit
Similar (b)
Rabbit
Rabbit
Monkey
Rabbit
R a bb i t
Rabbit
Rabbit
Rabbit
Rabbit
Monkey
(1969)
C 1969)
From Green et al. (1978)
(a) Response of the rabbit to test agent instilled with
a corneal applicator more closely approximated the
monkey response.
(b) Peak effects were observed in rabbits at 7 days and
in monkeys at 8 to 12 days.
(C) In some cases, the two species showed similar effects.
Table 2 :
Comparison of Re8ponse of the Rabbit and Monkey Eye
to Irritants.
More
Sensitive
Species
Rabbit (a)
Test Agent
Surf actant
formulations
1% Sodium hydroxide
Cytarabi ne
hydrochloride
Liquid detergent
Various materials
5% Soap solution
Detergent
Chloroacetophenone
Iodine solution
Surfactant formulations
Commercial shampoos,
cationic detergents
Variety of substances
5% Sulfuric acid
Reference
Buehler and Newmann
(1964)
Buehler and Newmann
(1964)
Elliot and Schut
(1965)
Beckley (1965)
Carter and Griffith
(1965)
Beckl.ey et al.
Beckley et al.
MacLeod (1969)
Hood et al. (1971)
Benke et al. (1977)
Gershbein and
McDonald (1977)
Green et al. (1978)
Green et al. (1978)
Cc)
—14—
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Table 3 . Eye Scoring Scheme Based on the Use of a
Slit Lamp Biomicroscope and Fluorescein.
“Accept “Probably Injur —
Site “Accept” with Caution” bus to Human
Eyes”
Conjunctiva Hyperemia Chemosis, less Chernosis, greater
without than 1 mm at than 1 mm at the
chemosis the limbus limbus
Cornea Staining, Confluence (b) Staining with
corneal of staining at infiltration or
stippling (a) 24 to 48 hours edema
without con-
fluence at
24 ? ours
Anterior Flare Cc) (visibil—
Chamber 0 0 ity of slit beam).
Rubeosis of iris
Ta) Corneal stippling: multiple discrete punctate irregular-
ities in the corneal epithelial layer which retain fluorescein.
(b) Confluence: uniform zones for fluorescein retention larger
than 1 mm in diameter.
(C) Flare: Tyndall effect in a beam traversing the aqueous
humor.
The EPA guidelines for labeling are listed (Table 4,
Campt 1981, Federal Register , 49, #188, 1984).
—15—
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Table 4 :
Label Statements Regarding Eye Irritation Hazards
Due to Pesticides.
I
Corrosive;
(irrever-
sible de-
struction
of ocular
tissue)
or corneal
involve-
ment or
irritation
persist ing
for more
than 21
days
Corrosive. *
Causes irre-
versible eye
damage. Harm-
ful if swal-
lowed. Do not
get in eyes or
on clothing.
Wear (goggles,
face shield,
or safety
glasses) **
Wash thoroughly
with soap and
water after
handling. Re-
move contamin-
ated clothing &
wash before
reuse.
If in eyes :
Flush with
plenty of
water. Get
medical
attention.
If swallowed :
drink promptly
a large quan-
tity of milk,
egg whites,
gelatin solu-
tion, or, if
these are not
available,
drink large
quantities of
water. Avoid
alcohol. NOTE
TO PHySICIAN :
Probable
mucosal damage
may contra-
indicate the
use of gastric
lavage.
Skull and
Crossbones
Toxicity
Signal
& “Poison”
Precautionary
Practical
Category
Word
Required
Statement
Treatment
Danger
No
* The term “corrosive” may be omitted if the product is not
actually corrosive.
** Choose appropriate form of eye protection. Recommendation
for goggles or face shield is more appropriate for indus-
trial, commercial, or nondoinestic uses. Safety glasses may
be recommended for domestic or residential use.
—16—
-------
(cont ‘d)
Table 4 :
II
Corneal
involve-
ment or
irritation
clearing
in 21 days
or less
Causes sub-
stantial but
temporary eye
injury. Do not
get into eyes
or on clothing.
Wear (goggles,
face shield, or
safety glasses.
Harmful if
swallowed.
Wash thoroughly
with soap and
water after
handling.
emove contam-
inated clothing
and wash before
reuse.
Same as above;
omit NOTE TO
PHYSICIAN
statement.
** Choose appropriate form of eye protection. Recommendation
for goggles or face shield is more appropriate for indus-
trial, commercial, or nondomestic uses. Safety glasses may
be recommended for domestic or residential use .
Skull and
Crossbones
Toxicity
Signal
& ‘ t Poison”
Precautionary
Practical
Category
Word
Required
Statement
Treatment
NO
Warning
-17—
-------
Table 4 : (cont’d)
Toxicity
Category
Signal
Word
Skull and
Crossbones
& “Poison”
Required
Precautionary
Statement
Practical
Treatment
III
Corneal
involve-
ment or
irritation
clearing
in 7 days
or less
Caution
No
Causes
(moderate) eye
injury (irrita-
tion). Avoid
contact with
eyes or cloth—
ing. Wash
thoroughly with
soap and water
after handling.
If in eyes:
Flush with
plenty of
water. Get
medical
attention if
irritation
persists.
Iv
Minimal
e ffects
clearing
in less
than 24
hours
Caution
No
None required.
None required.
—2.8—
-------
Figure ii !ys Matomy
A. Paipebral Conjunctiva
B. Forniz
C. aui sr Conjunctive
D. Liabus
E. Cornea] £pithsliu
F. D*scsmst’i M. brans
a. Endothetiua
M. Corneal StroLa
I. Cilia
J. Tarsal Plate
K.
L.
14.
N.
0.
P.
a.
R.
S.
1’.
Meibomlan Gland
Orbicul.aris Nembran.
Iris
Anterior a’ta.b.r
Posterior C) aaber
lAne
Chary Bodies
Zorni lee
Retina
Choroid
U. Selera
V. Optic
pap Lila.
W. Optic
nerve.
X. Niettat—
ing 5 . -
bran..
—19—
-------
Figure 2 : Proposed Testing Scheme.
Chemicals to
be tested
‘Er
pH measurement pH < 2 or > 11.5
pH > 2 and < 11.5
in vitro
tests ____ positive tests
Negative or No
equivocal test further
testing
Dermal Severely
irritation irritating
test to corrosive —
Nonirritat ing
to moderately
irritating
Rabbit eye test
—20—
-------
Definitions For Eye Irritation Testing
Irritation : The production of reversible changes in the
ocular structures following the application
of a test substance to the anterior structure
of the eye.
Corrosion : The production of irreversible changes in the
ocular structures following the application
of a test substance to the anterior structure
of the eye.
Bulbar conjunctiva :
The mucous membrane loosely attached to the
orbital septum, underlying anterior sciera,
and Tenan’s capsule except at the limbus,
where the conjunctiva fuses with Tenan’s
capsule for 3 mm.
Palpebral conjunctiva :
Lines the posterior surface of the eyelid,
firmly attached to the underlying tarsus, and
attaches to the sclera to become the bulbar
con j unct iva.
Chemosis : Conjunctival swelling.
Blepharitis : The inflammation of the eyelids.
Blepharospasm : Twitching of the eyelids.
Anterior chamber : The space filled with aqueous humor bounded
anteriorly by the cornea and posteriorly by
the iris.
Injection : Congestion of blood vessels, a term synonymous
with hyperemia.
jyphema : A term for blood in the anterior chamber.
Limbus : Junction of the cornea with the sciera.
Pannus : The infiltration of the cornea by blood
vessels.
Sciera : The external white layer of the eye composed
of fibrous tissue.
—21—
-------
tiveal tract : Composed of the iris, ciliary body, and choroid.
The ciliary body is composed of the ciliary
processes which make the aqueous humor and the
ciliary muscle which functions in accommodation.
The iris and ciliary body are called the anterior
area and the terms anterior uveitis, iritis,
and iridocylitis are synonymous.
Choroid : The posterior portion of the uveal tract and
the middle tissue of the eye between the sciera
and retina. The terms posterior uveitis and
choroiditis are synonymous.
Uveitis : A general term for inflammatory disorders of
the uveal tract and may involve one or all
three portions simultaneously.
Pachometer : An instrument used to measure corneal thickness.
Tonorneter : An instrument for measuring intraocular pressure.
—22—
-------
Bibliography
Baldwin, H.A., McDonald, T.O., Beasley, C.I3. (1973). Slit
Examination Of Experimental Animal Eyes. II. Grading Scales
And Photographic Evaluation Of Induced Apthological Conditions.
J. Soc. Cosmet. Chezn . 24:181—195.
Ballantyne, B., Swanston, D.W. (1972). Ocular Irritation Tests
(1972). Br. J.Pharmacol . 46: 577—578.
Balls, N. and Homer, S.A. (1985). The FRAME Interlaboratory
Program In Vitro Cytotoxicity. Fd. Cheni. Toxic . 23: 209-213.
Bosshard, E. (1985). Review On Skin And Mucous Membrane
Irritation Tests And Their Application. Fd. Chem. Toxic. ,
23: 149—154.
Beckley, J.H. (1965). Comparative Eye Testing; Man Vs. Animal.
Toxicol. Appi. pharmacol . 7: 93-101.
Beckley, 3.H., Russell, T. ., Rubin, L.F. (1969). use Of The
Rhesus Monkey For Predicting Human Response To Eye Irritants.
Toxicol. Appi. Pharmacol . 15: 1—9.
Benke, G.M., Brown, N.M., Walsh, N.J., Drothmari, R.B. (1977).
Safety Testing Of Alkyl polyethoxylate Nonionic Surfactants.
I. Acute Effects. Fd. Cosmet. Toxicol. , 15 (5): 309—318.
Bloom, W., Faweet, D.W. (1975). In: Textbook Of Histology .
W.B. Saunders Co., Philadelphia, Pa., pp. 917—963.
Borenfreund, E., Shopsis, C., Barrero, 0., Sathe, S. (1983).
In Vitro Alternative Irritancy Assays: Compar2son Of Cytotoxic
and Membane Transport Effect Of Alcohols. Ann. N.Y. Acad .
Sci., 407: 416—419.
Buehler, E.V., Newman, E.A. (1964). A Comparison Of Eye
Irritation In Monkeys And Rabbits. Toxicol. Appi. pharmacol .
6: 701—710.
2urstein, N.L. (1980). Corneal Cytotoxicity Of Topically
Applied Drugs, Vehicles And Preservatives. Supp. Ophthalmol .
25 (1): 15—30.
Burton, A.B.G. (1972). A Method For The Objective Assessment
Of Eye Irritation. Fd. Cosmet. Toxicol . 10: 209—217.
campt, D.D. (1981). Letter, Label Improvement Methods: Change
In Test Methods For And Categorization Of Eye Irritation.
EPA. PR 81-3 .
—23—
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DUE TO CIRCUMSTANCES BEYOND OUR CONTROL,
PAGE 24 OF THIS REPORT IS MISSING.
ALL EFFORTS TO OBTAIN THE MISSING PACE
HAS BEEN UNSUCCESSFUL, WE ARE MAKING THE
REMAINER OF THIS REPORT AVAILABLE TO THE
PUBLIC
—24—
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Environmental Protection Agency (1981). Eye Irritation Testing.
An Assessment Of Methods And Guidelines For Testing Materials
For Eye Irritancy. EPA-560/l1—82—00i, Washington, D.C.
Estable, J.L. (1948). The Ocular Effect Of Several Irritant
Drugs Applied Directly To The Conjunctiva. Am. . Ophthalmol .
31: 837—844.
Food & Drug Administration, Illustrated Guide For Grading Eye
Irritation Byj azardous Substances . Washington, D.C., Government
Printing Office (1965).
Federal Register , Interagency Regulatory Liaison Group, 46 (14):
7075—7077 (1981).
Friedenwald, J.S., Hughes, W.F., Herrmann, H. (1944). Acid—base
Tolerance Of The Cornea. Arch. Ophthalmol . 31 (4): 279—283.
Friedenwald, J.W., Hughes, W.F., Herrman, H. (1946). Acid Burns
Of The Eye. Arch. Ophthalmol . 35: 98—108.
Gartner, S. (1944). Blood Vessels Of The Conjurictiva. Arch.
Ophthalmol . 36: 464—471.
Gasset, A.R., Ishil, Y. , Kaufman, N.E., Miller, T. (1974).
CytotOxicity Of Ophthalmic Preservatives. Am. . O thalmo1 .
78 (1): 98—105.
Gershbein, L.L., McDonald, J.E. (1977). Evaluation Of The
Corneal Irritancy On Test Shampoos And Detergents In Various
Animal Species. Fd. cosmet. Toxicol . 15: 131—134.
Goldberg, A.M. (1985). An Approach To The Development Of In
Vitro Toxicology Methods. Fd. Chem. Toxic . 23: 205—208.
Grant, M.W. (1974). Toxicology Of The Eye , 2nd edition,
Springfield, IL ., Thomas.
Green, W.R., Sullivan, 3.B., Hehir, R.M., Scharpf, L.F.,
Dickinson, A.W. (1978). A Systematic Comparison Of Chemically
Induced Eye Injury In The Albino Rabbit and Rhesus Monkey . The
Soap And Detergent Association, New York.
Griffith, J.A., Nizon, G.A., Bruce, R.D., Reer, P.J., Banaan,
E.A. (1980). Dose—response Studies With Chemical Irritants
In The Albino Rabbit Eye As A Basis For Selecting Optinwm
Testing Conditions For Predicting Hazard To The Human Eye,
Toxico].. &Appl. Pharmac . 55: 501—513.
—25—
-------
Hagiwara, H., and Sugiwia, S. (1953). The Use Of Caster-oil
And Tween 80 As An Ophthalmic Base. Acta Soc. Ophthalmol .
(Jap) 57: 1—5.
Hirst, L.A., Kenyon, ICR., Fogle, J.A., Hanninen, L., Stark,
W.J. (1981). Comparative Studies Of Corneal Surface Injury
In The Monkey And Rabbit. Arch. Ophthal . 99:1066—1073.
Holly, F.L, Lemp, M.A. (1971). Wettability And Wetting Of
Corneal Epithelium. Exp. Eye Res . 11: 239—250.
Hood, C.I., Gasset, A.R., Ellison, E.D., Kaufman, H.E. (1971).
The Corneal Reaction To Selected Chemical Agents In The Rabbit
And Squirrel Monkey. Amer. 3. Ophthalmol . 71: 1009-1017.
Hughes, W.F. (1946). Alakli Burns Of The Eye. 1. Review Of
the Literature A1?d Summary of Present Knowledge. Arch. Ophth—
almol . 34: 423—449.
Jampol, L.M., Newfe]d, A.H., Sears, M.L. (1975). Pathways
For The Response Of The Eye To Injury. Invest. Ophthalmol .
14: 184—189.
Kemp, R.B., Meridith, R.W., Gamble, S.H. (1985). Toxicity Of
Commercial Products On Cells In Suspension Culture: A Possible
Screen For The Draize Eye Irritation Test. Ed. Chem. Toxic .
23: 267—270.
Krueger, R. (1959). Experinientelle Und Klinische Beobachtungen
Zur Beharidlund Der Alkaliveratzung Mit Ascorbinsaure. Bericht
p. Phthalm. Ges . 62: 255—258.
Leighton, 3., Nassauer, 3. and Tchao, R. (1985). The Chick
Embryo In Toxicology: An Alternative To The Rabbit Eye: Pd.
Chem. Toxic . 23: 293—298.
Leopold, I.H. (1945). Local Effects Of Detergents On Ocular
Structures. Arch. Ophthalmol . 34: 99—102.
Lewin, L. and Gui].lery, H. (1913). Die Wirkungen Von Arznei—
mittein Und Giften Auf Das Age , 2nd ed. Berlin: Hirschwald.
Luepke, N.P. (1985), Hen’s Chorioallantoic Membrane Test For
Irritation Potential. Fd. Chem. Toxic . 23: 287—291.
MacLeod, I.F. (1969). Chemical Mace; Ocular Effects In Rabbits
And Monkeys. 3. Forensic Sci . 14: (1) 34—47.
Mann, I., Pullinger, B.D. (1942). A Study Of Mustard Gas
Lesions Of The Eyes Of Rabbits And Men. Proc. R. Soc. Med .
35: 29—244.
—26—
-------
Marsh, R.3., Maurice, D.M. (1971). The Influence Of Non—ionic
Detergents And Other Surfactants On Human Corneal Permeability.
Exp. Eye Res. , 11:43—48.
Marzulli, F.N. (1968). Ocular Side Effects Of Drugs. Fd.
Chem. Toxic. , 6: 221—234.
Marzul].i, F.N., Simon, M.E. (1971). Eye Irritation From
Topically Applied Drugs And Cosmetics: Preclinical Studies.
Am. 3. Optom . 48: 61—79.
Maul, E., Sears, M.L. (1976). Objective Evaluation Of Experi-
mental Ocular Irritation. Invest. Ophthalmol . 15 (4).
Maurice, D.M. and Ciardini, A.A. (1951). A Simple Optical
Apparatus For Measuring The Corneal Thickness, And The Average
Thickness Of The,Human Cornea. Brit. 3. Ophth . 35: 169—177.
Maurice, D.M. (1968). Cellular Membrane Activity In The Corneal
Endotheliuni Of The Intact Eye. Experimentia . 24: 1094—1095.
McDonald, T.O., Baldwin, H.A., Beasely, C.H. (1973). Slit
Lamp Examination Of Experimental Eyes. I. Technique Of Illumin-
ation And The Normal Animal Eye. 3. Soc. Cosmet. Chem . 24:
163—180.
McDonald, T.O., Shadduck, J.A. (1977). Eye Irritation. In:
Advances In Modern Toxicology , Marzulli, F.N. and Maiback,
H.I. (eds)., vol. 4 Hemisphere, Washington and London.
McDonald, T.O., Seabaugh, V.M., Shadduck, 3.A., Edeihauser,
H.F. (1983). Eye Irritation. In: Derinato—toxicology , 2nd edition,
Marzulli, F.N., and Maiback, H.I. (eds). Hemisphere, Washington.
McLaughlin, R.S. (1946). Chemical Burns Of The Human Cornea.
Amer. J. Qphthalmol . 29 (11): 1355—1362.
Mishima, S., Hedbys, B.O. (1968). Measurements Of Corneal
Thickness with The Haig—Streit pachometer, Arch. Ophthalmol .
80: 710.
Muir, C.K., Flower, C., Van Abbe, N.J. (1983). A Novel Approach
To The Search For In Vitro Alternatives To In Vivo Eye Irritancy
Testing. Tox. Letters 18, 1—5.
Murphy, 3.3. (1986). Memorandum. Trip Report For OECD Ad Hoc
Meeting On Acute Toxicity Testing. Paris, France, U.S. EPA.
—27—
-------
National Academy Of Sciences - National Research Council (1975).
In: Principles For Evaluating Chemicals In The Environment .
WAS Publication For The Environmental Protection Agency: Eye
Irritation, 104—105.
National Academy Of Sciences — National Research Council,
(1977). In: Principles And Procedures For Evaluating The
Toxicity Of Household Substances , Prepared By Committee For
The Revision Of NAS Publication 1138 for the Consumer Product
Safety Commission. ! ye Irritation , 41—54.
Organization For Economic Cooperation And Development (OECD)
(1986). Acute Eye Irritation/Corrosion , OECD Publications And
Information Center, Washington, D.C.
Parish, W.E. (1985). Ability Of In Vitro Tests To Represent
Histopathological Features Of Acute Eye Inflammation. Fd.
Chem. Toxic . 23: 215—227.
Phister, R.R. (1973). The Normal Surface of Corneal Epithelium:
A Scanning Electron Microscopic Study. Invest. Ophthalmol .
12: 654—668.
Potts, A.M., Gonasun, L.M. (1975). Toxicology Of The Eye. In:
Toxicology — The Basic Science Of Poisons . Casarett, L.3. and
Doull, 3. (eds). Macmillan Publishing Co., Inc., N.Y., N.Y.,
275—309.
Prince, J.H., Dieseni, C.D., Eglitis, I., Ruskell, G.L. (1960)
Anatomy And Histology Of The Eye And Orbit In Domestic Animals .
Springfield, IL: Thomas.
Reinhardt, C.A., Pelli, D.A., Eglitis, I., Ruskell, G.L. (1985).
Interpretation Of Cell Toxicity Data For The Estimation Of
Potential Irritation. Fd. Chem. Toxic . 23: 247—252.
Seabaugh, V.M., Osterberg, R., Hoheisel, C.H., Murphy, 3.,
Bierbower, C. (1976). A Comparative Study Of Rabbit Ocular
Reaction To Various Exposure Times To Chemicals, Toxicology
And Appl. Pharm . Vol. 37, No. 1.
Sciafe, Tl.C. (1985). An In Vitro Cytotoxicity Test To Predict
The Ocular Irritation Potential Of Detergents And Detergent
Products. Fd. Chem. Toxic. , 23: 253—258.
Shopsis, C., Borenfreund, E., Walberg, 3., Stark, D.M. (1985).
A Battery Of Potential Alternatives To The Draize Test. Uridine
Uptake Inhibition, Morphological Cytotoxicity Macrophage Chemo-
toxic, And Exfoliate Cytology. Fd. Chem. Toxic. , 23: 259—266.
—28—
-------
Shuster, 3., Kaufman, H.E. (1974) Letter, Invest Ophthalmol. ,
13: 892—893.
Sugar, A. (1979). Clinical Specular Microscopy. Surv. Ophthalrnol .
24 (1): 21—32.
Swanston, D.W. (1985). Clinical Specular Microscopy. Surv.
Ophthalmol . 23: 169—173.
Van Abbe, N.Y. (1973). Eye Irritation: Studies Relating To
Responses In Man And Laboratory Animals. 3. Soc. Cosinet. Chetn .
24: 685—692.
Walton, R.M., Heywood, R. (1978). Applanation Tonometry In
Assessment Of Eye Irritation. 3. Soc. Cosmet. Chem . 29: (6):
365—368.
Weil, C.S., Scala, R.A. (1971). Study Of Intra— And Interlabor—
atory Variability In The Results Of Rabbit Eye And Skin Irrita-
tion Tests. Toxicol. Appi. pharmacol . 19: 276-319.
Weitman, A.S., Sparber, S.B., 3urtshuk, T. (1965). Comparative
Evaluation And The Influence Of Various Factors On Eye Irritation
Scores. Toxicol. Appi. pharmacol . 7: 308—319.
Williams, S.J., Graysel, G.J., Kennedy, G.L. (1982). Evaluation
of Ocular Irritancy Potential: Intralaboratory variability And
Effect Of Dosage Volume. Toxicol. Lett . 12, 235.
Williams, s.c. (1985). changing Concepts Of Ocular Irritation
Evaluation: pitfalls And Progress. Fd. Chem. Toxic . 23: 186—193.
—29—
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