In vitro test for ocular toxic properties

A reagent and method for in vitro determination of the eye irritating properties of individual compounds and/or mixtures is disclosed. The material to be assessed is combined with the reagent which provides a response, for example, the production of a precipitate, which is proportional to the deleterious response elicited by the material in the human eye.

TECHNICAL FIELD 
This invention relates to the field of testing materials for their capacity 
to irritate the human eye. More specifically, the invention relates to an 
in vitro test which can predict the ability of a specified material to 
cause temporary or permanent damage if placed in contact with the human 
eye. 
BACKGROUND ART 
In the United States and elsewhere in the technologically developed world, 
it is customary and often legally mandated, to assess the capacity of 
consumer products such as shampoos, detergents, cosmetics, or other 
materials which are likely to be handled by the general public without eye 
protection, to cause temporary or permanent damage to the human eye. 
Because of the sensitivity and criticality of ocular tissue, it is clearly 
desirable and necessary to provide adequate labeling in the case of mild 
and temporary eye irritants or even to restrict the commercialization of 
compositions which contain components extremely damaging to the eye. It is 
estimated that more than a million tests are performed annually in the 
United States to examine various cosmetic and household produts for their 
potential harmfulness to the human eye. As in any instance where large 
numbers of tests need to be performed to screen for a particular property, 
it is desirable to have a testing procedure which is rapid, inexpensive, 
and reliable. Certainly an in vitro test would be most desirable. 
Such an in vitro test is not currently available for substances which might 
irritate the eye. The most commonly utilized current screening procedure 
is an in vivo one, the Draize rabbit-eye test (Draize, J. H., et al J 
Pharmacol Exptl Therap (1944) 82:377). The disadvantages of this test are 
legion. First, as it is an in vivo procedure it necessarily involves some 
degree of maltreatment of test animals, and considerable expense. 
Secondly, it is lengthy. The procedure consists of placing the substance 
to be tested into the eyes of 3-9 albino rabbits and scoring the degree of 
irritation on the conjunctiva, cornea, and iris after periods of 3-21 
days. The scoring of the results is, of course, subjective to some degree. 
The individuality of the test animals makes the results inherently 
unreproducible. Many attempts have been made to refine and improve the 
Draize test and to minimize its disadvantages (see, for example, Batista, 
S. P., et al, Soc Cosmet Chemists (1965) 16:119; Kay, J. H., et al, Am 
Perfumer Cosmetics (1965) 80:61; Gaunt, I. F., et al, J Soc Cosmet 
Chemists (1964) 15:209). However, the inherent deficiencies of this 
testing approach make impossible the attainment of the objective of a 
cheap, reliable, reproducible and predictive test for eye irritating 
properties. 
As in all animal tests, the correlation with effects of the materials in 
human subjects is imperfect; however, the Draize test has become 
sufficiently well recognized, that a more straightforward test which 
produces results identical to those of the Draize would represent an 
improvement in the state of the art, and correlation with Draize results 
can reasonably be used to evaluate such a test. 
The clear desirability of an in vitro procedure has led a number of 
researchers to devise tests involving cell cultures as opposed to whole 
animals. For example, the method of Ferguson, T. F. M., et al, Food and 
Cosmet Toxicol (1974) 12:359 employs cultured mouse fibroblast cells and 
uses the ability of a material to inhibit the uptake of tritiated uridine 
by these cells as a measure of its eye irritation capacity. The method of 
Stark, L., et al, Chemical Week (1983) May 26:27 uses a procedure whereby 
mouse cell cultures are exposed to irritants, and the fluids from such 
cultures evaluated for their effect on the migration of macrophages. The 
method of Char Jumblatt, M., Vision Res (1981) 21:45 uses the level of 
plasminogen activator in rabbit corneal cells in culture as a measure of 
the eye-irritating capability of a substance. Finally, inhibition of 
culture growth in fibroblast or HeLa cells was used as a criterion by 
Litterst, C. L., et al, Arch Environ Health (1971) 22:454. Two additional 
methods employ mouse or rabbit ileum and assess, respectively, the 
penetration capacity of the substance to be tested (Muir, C. K., 
Toxicology Letters (1983) 19:309) and the response of a chorioallantroic 
membrane of chick embryos (Leighton, J., et al, Proc of the Symposium, 
Product Safety Evaluation, A. M. Goldberg, Editor, Mary Ann Liebert 
Publications, New York (1983)). 
All of the foregoing in vitro methods require either living cells in tissue 
culture or isolated membranes in tissue culture . All suffer from the 
disadvantages of lack of reproducibility, lack of objectivity, and lack of 
perfect correlation with the property desired to be measured, as does the 
Draize method. While these procedures provide an alternative to the 
strictly in vivo approach of Draize, they do not achieve the simplicity 
and standardization one expects from testing based on chemical reagents 
which can be manufactured and stored, and reproduced exactly. The method 
of the present invention offers such a test. It provides a standard, 
quick, reproducible, objective measure of the capacity of any material to 
cause irritation in the human eye. It does not involve use of small 
animals, and does not require the expense of maintaining, caging, and 
feeding facilities for them. 
DISCLOSURE OF THE INVENTION 
The invention provides a reagent and a method for assessing the capacity of 
materials to cause temporary or permanent irritation in the human eye. The 
magnitude of the response given by the reagent test system of the 
invention correlates to the severity of the eye irritation which will be 
caused by the tested material. The reagent is a defined or semi-defined 
mixture of materials which has the advantages and properties of standard 
chemical reagents. The procedure is straightforward and rapid. The reagent 
and substance to be tested can be mixed and the result assessed visually 
without instrumentation, or can be quantitated, using a variety of 
laboratory instruments which are commonly available in analytical 
laboratories, if desired. 
Accordingly, in one aspect, the invention relates to a reagent useful for 
predicting the toxicity of a material to the human eye wherein the reagent 
comprises a mixture of proteinaceous substances which are capable of 
quantitative response to the presence of an ocular irritant. In other 
aspects, the invention relates to methods of predicting the ocular 
toxicity of materials using the reagent of the invention and to test kits 
useful in performing such methods. 
MODES OF CARRYING OUT THE INVENTION 
A. Definitions 
As used herein, "toxicity" of a material to the human eye or "ocular 
toxicity" refers to the ability of this material to cause negative 
responses in the human eye which take the form of either temporary or 
permanent damage to its tissues. This toxicity is evidenced by causing 
pain, opaqueness of the cornea or iris or both, congestion, swelling, 
hemorrhaging, or gross destruction of the iris, redness or dilation of the 
conjunctiva or production of discharge. Thus, the word "toxicity" or 
"toxic" in this context is defined broadly to include any discomfort or 
injury which results from the presence of a material in contact with the 
human (or other mammalian) eye. 
"Clear aqueous liquid" refers to a liquid, usually a mixture, which is, in 
substantial part, water and which is functionally transparent to light in 
the visible range. "Functionally transparent to light in the visible 
range" means that sufficient light is transmitted by the sample to permit 
measurable absorbances to be detected upon precipitation of the components 
of the liquid. For example, even absorption of visible wavelengths in 
amounts of about 0.8 absorbance units in a 1 cm cuvette permits a readable 
range for additional absorbance caused by such precipitation. Of course, 
the permissible absorbance of such functionally transparent samples 
depends on the path length provided and upon the absorbance range 
measurable by the available instrument. 
"Ocular irritant" refers to a substance which is capable of displaying 
toxicity to the human eye when placed in contact with it. 
"Compatible" conditions of pH and/or ionic strength refers to ranges of 
these parameters which are consistent with the property of the reagent to 
precipitate only in the presence of an ocular irritant. 
"Reagent" refers to the reaction mixture in contact with the sample to be 
tested; "preparation" refers to a material which, when diluted with 
solvent and sample results in the reagent. 
B. General Description 
B.1 General Parameters of the Testing Procedure 
The present invention provides a reagent which, when mixed with a substance 
to be tested for ocular toxicity, produces a response which is measureable 
qualitatively or quantitatively in proportion to the ocular toxicity of 
the sample. The direct response of mixing the sample with the reagent is 
the formation of a precipitate, whose magnitude can be assessed using a 
variety of techniques which are described in detail below. The formation 
of the precipitate is, in a very rough sense, a mimetic response to that 
produced in the eye by the irritant. Therefore, the response of the 
reagent to the substance to be tested can be used as a predictor of the 
response of eye tissue to the same material. 
B.2 The Reagent 
The reagent is used in the form of a clear aqueous liquid which will be 
mixed with the substance to be tested. However, the active ingredients of 
the reagent mixtures of these substances can be prepared as solids, and 
when subsequently dissolved to form a clear aqueous liquid reagent, will 
precipitate or otherwise respond to the presence of an ocular irritant. 
Thus, the mixture of components in the desired amounts can be supplied not 
only already dissolved to form the finished reagent, but also in solid 
form either as a powder, lyophilized solid, or a gel, which can be 
subsequently reconstituted to form the reagent. 
In any case, the reagent is preferably supplied in a somewhat more 
concentrated form than the final concentration of components desired, so 
that it can be added to a reaction mixture with a diluent and with the 
sample to be tested. 
The mixture itself is a composition of proteinaceous materials, amino 
acids, carbohydrates, and ionic compounds which, in some sense, mimics the 
response of human eye tissue to contact with materials to be tested. 
The clear aqueous liquid reagent of the invention contains solutes or 
colloidal particles which will remain in solution or in a colloidal state 
until a ocular irritant is added, whereupon precipitation occurs. To 
achieve this, the reagent contains at least one precipitant and at least 
one stabilizer, and is maintained at compatible pH and ionic strength 
conditions. It is preferable to include at least one enhancer, and to 
protect the reagent against deterioration by supplying antibodies and 
enzyme inhibitors. 
Precipitants represent the desired response of the reagent to an ocular 
irritant--i.e. these materials precipitate to give turbidity or a 
separable solid from the mixture. Effective precipitants include the 
globular proteins, which are best employed as mixtures of several 
different globulins such as globulin G.sub.1, G.sub.2 and G.sub.3 or 
subcombinations thereof. A single globulin is operable, though not as 
sensitive. The total globulin concentration in the finished reagent is in 
the range of 0.001-10%, preferably 0.01-5%, depending on the class of 
globulins used. Alternate precipitants suitable for the reagent of the 
invention include, for example, macroglobulins, certain 
glycosaminoglycans, and mucoproteins. 
(In the above paragraph, and in those following, the concentration ranges 
are given as wt/volume percentages (unless otherwise specifically 
indicated), and reference the volume of finished reagent. In typical 
preparations, the concentration will be 5-10 times higher, so as to permit 
dilution as outlined above.) 
Stabilizers prevent premature aggregation of the precipitant and may make 
the extent and form of the aggregation more reproducible. Suitable 
stabilizers include, for example, amino acids, such as glycine, glutamine, 
valine, leucine and the like, peptides of 200-5000 daltons and 
non-globulin proteins such as albumins. A wide range of concentrations and 
of combinations of stabilizers is workable. In one preferred embodiment, 
glycine may be used as the only stabilizer in the concentration range 
about 0.005-0.5%. In general, total stabilizer concentration is in the 
range of 0.001%-10%, preferably 0.1%-5% depending on the nature of the 
stabilizer chosen. 
Compatible pH range and ionic strength may be maintained by adjusting the 
buffering capacity and ionic status of the foregoing two required 
components--i.e. precipitant(s) and stabilizer(s) or, preferably are 
obtained by providing suitable ionic compounds or buffers. A compatible pH 
range is between about 1-10, but preferably between about 2-9. Higher pH 
values denature the precipitant and allow it to remain in solution, even 
in the presence of ocular irritants, lower pH values may cause premature 
precipitation. Suitable buffers in this range include phosphate salts, 
acetate salts, Tris-Cl, bicarbonate and a variety of other compounds known 
in the art. Ionic strength can vary over a wide range from about 0.05 M to 
0.5 M, and is generally high enough to be workable (due to the presence of 
charged moieties in the precipitant and stabilizer) even in the absence of 
additional salts. However, this parameter can be increased, if desired, by 
the addition of such commonly available salts as NaCl, KCl, or NaNO.sub.3. 
It is desirable, though not absolutely necessary, to include in the reagent 
enhancers which interact with the molecules of precipitant so as to 
increase its aggregation in the presence of ocular irritants. Such 
enhancers are, most typically, glycoproteins such as ovomucoids, 
mucopolysaccharides; mucin and carbohydrates such as glucose; and lipids, 
such as phospholipids. The desired concentration range varies, of course, 
with the nature of the enhancer but is generally in the range of 0-10%. 
The reagent may be further protected from deterioration by addition of 
bacteriostats or bacteriocides such as sodium azide, and by use of enzyme 
inhibitors, such as N-ethylmaleimide. 
In the foregoing paragraphs, applicants have set forth the parameters 
required to constitute the reagent as a defined medium using predetermined 
amounts of available substances. However, it is also possible to achieve 
the result of a workable reagent, containing the proper concentrations of 
precipitant and stabilizers, and, indeed, suitable amounts of optional 
components such as enhancer by utilizing extracts of natural materials 
which contain, for example, globulins, peptides, albumin, and other 
desired components of the reagent. Two natural materials which are 
particularly preferred to provide such extracts are egg whites, jack beans 
and other legumes and combinations, thereof all of which contain 
globulins. The preparations can be made using water or salt solutions as a 
diluent, however, they are preferably made using an extracting solution 
containing, for example, ionic compounds and buffers, and/or supplementary 
components such as EDTA to adjust the concentrations of ions which affect 
the precipitant. The extracting solution, if such natural materials are 
used, may not need to contain precipitants or stabilizers, as these are 
found in the natural materials themselves. 
B.3 The Method 
A major virtue of the method of the invention is its simplicity. The crux 
of the procedure is merely contacting the reagent of the invention with 
the material to be tested. The results can be made quantitative either by 
quantitative measurement of the amount of precipitate directly, by 
indirect measurement of the amount of precipitate by, for example, 
colorimetric means, or by ascertaining the presence or absence of a preset 
quantitative response using serial dilutions of sample in admixture with 
the reagent. 
The mass of the precipitate can be obtained by separation from the mixture, 
e.g., by centrifugation, drying and weighing. However, more preferred is 
measurement of turbidity, which method is more rapid and less subject to 
complications due to physical variation of the precipitate during drying. 
The amount of turbidity formed can be measured using standard absorbance 
readings obtained with spectrophotometers or colorimeters. It is important 
to use both the reagent and sample as "blank"s in such procedures, as some 
light absorbance or scattering will be caused by the presence of 
macromolecular species in the reagent itself or by the sample. In some 
samples, the total absorbance contributed by the sample and reagent 
separately may be on the order of 0.8 OD units or more per cm path length, 
so that it may be desirable to decrease the path length if absorbance 
measurements are used, or to employ a dedicated instrument calibrated to a 
higher absorbance range. Of course, the absorbance of both the sample and 
reagent blank are subtracted from that of the test sample. Light 
scattering due to turbidity can best be quantitated at 340 nm or 430 nm. 
Resolution to a narrow wavelength band is unnecessary. 
In the alternative, nephelometry may be substituted for absorbance 
readings, and, indeed, may be preferred in some cases. This method of 
quantitation is, in general more sensitive and covers a larger range of 
concentrations than absorbance measurements. However, due to the 
ubiquitous use of colorimetry, this method is preferred for most 
commercial applications. 
The amount of precipitate may also be quantitated by measuring the amount 
of protein. The precipitant is separated from the mixture by, for example, 
centrifugation and removal of the supernatant, and the isolated 
precipitate subjected to standard techniques for protein determination 
known in the art, such as the buiret method, the method of Lowry (Lowry, 
O. H. et al, J Biol Chem (1957) 195:265) or by separation on 
polyacrylamide gel and staining a diagnostic band or bands with Coomassie 
blue or silver stain. 
Alternatively, the amount of precipitate can be assessed quantitatively by 
attaching a label to a component of the reagent so that the quantity of 
material carried down with the precipitate can be assessed by the quantity 
of label present in the precipitate or remaining in the supernatant. The 
label may be, for example, a radioactive material, a chromophore, or a 
fluorophore. In any case, the supernatant and precipitate are separated, 
and the amount of label read in the desired fraction by means appropriate 
to the nature of the label. 
If the label in the precipitate is quantitated, the amount present is a 
direct measure of the ocular toxicity of the sample; if the label is 
quantitated in the supernatant, the amount is inversely proportional to 
the toxicity. 
The proteinaceous precipitate can readily be labeled by a variety of means 
employing such commonly available .beta.-emitting radioisotopes as .sup.14 
C and .sup.3 H. The activity in the precipitate can then be determined by 
use of a Geiger counter, or that in the supernatant using scintillation 
counting. Other isotopes such as .sup.125 I or .sup.131 I can also be 
conjugated to the protein precipitant and counted using similar 
techniques. 
Conjugated chromophores and fluorophores are most conveniently determined 
in the supernatant fraction, although they may also be solubilized from 
the separated precipitate and measured. Convenient fluorophores include 
dansyl, fluorescein and rhodamine dyes. Convenient chromophores include 
for example p-nitroaniline, which absorbs at 405 nm. 
The label may also be a substrate for an enzyme catalyzed reaction which 
can be quantitated using standard means. For example, any of the 
substrates of the peroxidase reaction could be used as label, and the 
enzyme added to the supernatant fraction after separation of the 
precipitate to assess the concentration of substrate remaining. 
B.4 Evaluation of Results 
As set forth above, it would be desireable to have a perfect correlation 
between the results of the test as performed by the method of invention 
and the capacity of a material to irritate the human eye. However, the 
results of human experience with respect to the large number of irritants 
for which testing is desireable are not readily available in retrieveable 
form. The results of Draize testing on large numbers of substances is. 
Therefore, a threshold criterion for predictive validity of the testing 
procedures of the invention is correlation of its results with those of 
Draize testing on the same substances. 
Accordingly, calibration curves have been prepared which show the 
relationship between the absorbance readings obtained using 340 nm in 
assessing the turbidity caused by the test substances in the method of the 
present invention with the results of the Draize test. Similar procedures 
may be used to calibrate other primary results criteria such as absorbance 
at other wavelengths, nephelometric readings, absorption due to color 
reagents, and radioactivity. 
Typically, it is found that 340 nm absorbance follows a linear pattern with 
increasing concentration of test substance but plateaus at a level 
characteristic of each individual material. This is expected behavior in 
absorbance measurements, and therefore, it is possible to establish the 
position on the absorbance curve being read for each material tested. 
Using those data, testing can be done in the range of linearity for a 
particular material, thus assuring that the readings for that material 
will fit into the general calibration curve for the test method. 
Once each substance to be tested is shown to be generating readings in the 
linear range, by verifying the readings for, e.g., sample sizes of 100 
.mu.l, 200 .mu.l, and 500 .mu.l in a 1 ml total volume, the absorbances 
obtained at these sample sizes can be classified so as to correlate with 
the Draize results test ranges. A more detailed explanation of the 
specific correlations established using 340 nm absorption as the primary 
test criterion is given in C.2.b.

C. Examples 
The following examples are intended to illustrate but not to limit the 
invention. 
C.1 Preparation of Reagent Mixture 
The following are prepared by using a buffer solution in some cases 
containing plant or animal globulins to extract either egg white or jack 
bean powder or both. If egg white is used, the separated whites are 
diluted with the buffer using 1 ml egg white per 2 ml buffer. If jack bean 
powder is used, 2 g finely powdered bean is soaked in 100 ml extraction 
buffer for 2 hours, and filtered twice with Whatman #40 paper to remove 
residue. 
Preparations A-C are the concentrations of components shown in preparations 
which are typically diluted 2-10 times for the finished reagent 
concentration desired. 
Preparation A 
The first 16 components derive from the extraction buffer, the last 6 from 
jack beam powder. 
______________________________________ 
Compound Concentration 
______________________________________ 
CaCl.sub.2 0.02% 
KCl 0.04% 
MgSO.sub.4 0.01% 
NaH.sub.2 PO.sub.4 :H.sub.2 O 
0.01% 
NaCl 0.2 M 
isoleucine 0.002% 
glutamine 0.03% 
leucine 0.002% 
lysine:HCl 0.004% 
tyrosine 0.002% 
valine 0.002% 
NaOAc 0.1 M 
EDTA 0.1% 
N--ethylmaleimide 0.01% 
NaN.sub.3 0.02% 
glucose 0.1% 
globulin G.sub.1 0.1-0.2% 
Mucopolysaccharide 0.1-0.15% 
albumin 0.1-0.3% 
carbohydrates 0.2-0.3% 
lipids 0.3-0.5% 
saponins 0.001- 0.01% 
______________________________________ 
Preparation B 
The first 14 components derive from extraction buffer, the remaining 9 from 
egg white. 
______________________________________ 
Compound Concentration 
______________________________________ 
NaOAc 0.07 M 
NaCl 0.15 M 
EDTA 0.07% 
N--ethylmaleimide 0.07% 
NaN.sub.3 0.015% 
CaCl.sub.2 0.014% 
KCl 0.028% 
MgSO.sub.4 0.007% 
NaH.sub.2 PO.sub.4 :H.sub.2 O 
0.007% 
lysine:HCl 0.007% 
isoleucene 0.001% 
tyrosine 0.001% 
glutamine 0.021% 
valine 0.001% 
conalbumin 3% 
ovalbumin 22% 
lipids 0.4% 
carbohydrates 0.3% 
ovomucoid 4% 
globulin G.sub.1 0.5-1% 
globulin G.sub.2 0.5-2% 
globulin G.sub.3 0.2-2% 
glucose 0.1% 
______________________________________ 
Preparation C 
The first 16 components derive from extraction buffer, the last 10 from egg 
white and jack bean powder. 
______________________________________ 
Compound Concentration 
______________________________________ 
CaCl.sub.2 0.02% 
KCl 0.04% 
MgSO.sub.4 0.01% 
NaH.sub.2 PO.sub.4 :H.sub.2 O 
0.01% 
NaCl 0.15 M 
isoleucine 0.001% 
glutamine 0.02% 
leucine 0.001% 
lysine:HCl 0.002% 
tyrosine 0.001% 
valine 0.001% 
NaOAc 0.8 M 
EDTA 0.05% 
N--ethylmaleimide 0.1% 
NaN.sub.3 0.02% 
glucose 0.1% 
globulin G.sub.1 1-2% 
globulin G.sub.2 1-3% 
globulin G.sub.3 1-4% 
conalbumin 2% 
ovalbumin 5% 
ovomucoid 2% 
mucin 1% 
saponins 0.10% 
lipids 0.5% 
carbohydrates 0.5% 
______________________________________ 
C.2. Results 
C.2.a Preliminary Tests 
In one protocol, 200 .mu.l of preparation A were used in a total volume of 
1 ml. Serial sample amounts of 500 .mu.l, 250 .mu.l, 100 .mu.l and 50 
.mu.l were used, and optical density at 340 nm was read in a Beckman DV-8B 
spectrophotometer. The resulting absorbance was used as a criterion for 
ocular toxicity. Absorbances above about 1.0 OD unit were considered 
indicative of ocular toxicity. The results were placed in broad categories 
of: 
non-irritant (N) &lt;1.0 
mild irritant (Mi) 1.0-2.0 
moderate irritant (Mo) 2.0-2.5 
severe irritant (S) &gt;2.5 
The results in Table 1 below show absorbance values for 500 .mu.l sample 
size and the categorization of results using the arbitrary absorbance 
range criteria indicated. The categories are compared with those reported 
in standard tests as indicated. (The concentrations given in column 1 
refer to the concentration of the sample in the 500 .mu.l portion as added 
to the reagent mixture.) 
TABLE 1 
______________________________________ 
Invivo Draize 
Concentration/Sample 
OD.sub.340 
Class Test Results 
______________________________________ 
baby shampoo/100% 
0.63 N N (1) 
methyl paraben/0.2% 
0.75 N N (1) 
benzalkoniumchloride/1% 
2.51 Mo--S 
benzalkoniumchloride/0.5% 
2.43 Mo--S Mo--S (4) 
benzalkoniumchloride/0.1% 
2.43 Mo--S Mo (2) 
resorcinol/100% 
2.52 Mo Mo (4) 
resorcinol/5% 0.40 N N (4) 
sodium lauryl 1.00 Mi Mo--S (2) 
sulfate/40% 
propylene glycol/25% 
0.74 N N--Mi (3) 
thimerosal/2% 0.69 N Mi (4) 
thimerosal/0.5% 
0.002 N N (4) 
______________________________________ 
(1) Applied Biological Sciences, Glendale, CA. 
(2) Griffith, J. F., et al, Tox and Appl Pharmacol (1980) 55:501. 
(3) Conquet, T. H., et al, Tox and Appl Pharmacol (1977) 39:129. 
(4) Burstein, N. L., Survey of Opthamol (1980) 25:15. 
C.2.b Results of Calibrated Study 
The preliminary results obtained in C.2.a were further expanded by 
preparing correlation standards against Draize eye test results. 
First, an arbitrary scale of classifications correpsonding to the 
conventional use Draize classification was devised as set forth below. 
______________________________________ 
Rating Scale Draize Scale 
______________________________________ 
N 1.0 0 
N--Mi 1.5 1-10 
Mi 2.0 10-20 
Mi--Mo 2.5 20-40 
Mo 3.0 40-60 
Mo--S 3.5 60-80 
S 4.0 80-110 
______________________________________ 
Thus, for example, a test substance which received an average absorbance 
based scale rating of 3.5 would be classed as Mo-S; one which had an 
average scale value of 2.0 would be classed Mi. 
The classification average was obtained by averaging the class obtained at 
3 levels of sample volume, 50 .mu.l, 100 .mu.l, and 200 .mu.l in a 1 ml 
reaction mixture averaging these classes and rounding to the next highest. 
The class obtained for each sample volume was determined according to the 
scale set forth as follows. 
__________________________________________________________________________ 
Class 1.0 1.5 2.0 2.5 3.0 3.5 4.0 
__________________________________________________________________________ 
50 .mu.l 
0-0.4 
0.4-0.8 
0.8-1.2 
1.2-2.0 
2.0-2.5 
2.5-3.0 
3.0-3.5 
100 .mu.l 
0.4-0.8 
0.8-1.2 
1.2-2.0 
2.0-2.5 
2.5-3.0 
3.0-3.5 
&gt;3.5 
200 .mu.l 
0.8-1.2 
1.2-2.0 
2.0-2.5 
2.5-3.0 
3.0-3.5 
&gt;3.5 
&gt;3.5 
__________________________________________________________________________ 
Using these classes, the following results were obtained: 
______________________________________ 
50 .mu.l 
100 .mu.l 
200 .mu.l 
Ave Class 
______________________________________ 
5% thimerosal 
abs: 0.21 .29 .43 
scale: 1.0 1.0 1.0 1.0 N 
0.05% benzal- 
abs: 0.40 0.80 1.25 
koniumchloride 
scale: 1.5 1.5 1.5 1.5 N--Mi 
70% Isopropanol 
abs: 0.93 1.42 1.85 
scale: 2.0 2.0 1.5 2.0 Mi 
0.5% benzal- 
abs: 2.1 3.0 3.2 
koniumchloride 
scale: 3.0 3.0 3.0 3.0 Mo 
______________________________________ 
C.2.c Comparison of Draize and Invention Method 
Table 2 shows a comparison of results in the reagent of the invention using 
the calibrated system of C.2.b, the Draize rabbit eye test and general 
human experience. The results of the Draize test and their comparison to 
human experience was reported by Griffith, J. F., et al, Tox & Appl 
Pharmacol (1980) 55:501. 
______________________________________ 
Draize 
Human Eye 
Experience 
Test Preparation A 
______________________________________ 
Benzalkonium/ 
Mi--Mo Mo Mo--S 
chloride/0.1% 
Acetic acid/3% 
Mi--Mo Mo Mi--Mo 
Sodium lauryl/ 
N Mo N 
sulfate/10% 
Sodium lauryl/ 
Mi Mo Mi 
sulfate/29% 
NaClO.sub.2 Mi Mo Mi 
Formaldehyde/38% 
Mi S* (Inhibitor) 
Isopropanol/70% 
Mi Mo Mo 
______________________________________ 
*very severe 
In addition, 45 samples of household chemicals and common laboratory 
compounds, such as Ivory Liquid.RTM., Tide.RTM., acetone, sodium borate, 
cetalkonium chloride, thimerosal, and Chlorox.RTM., were tested using 
visual evaluation of results obtained when 500.mu.of test sample were 
added to 500.mu.l of Preparation B, above. Results were compared with 
those of the standard Draize test as reported in the literature, according 
to the classification noted above. The results were substantially 
identical in 35 of these trials. In another six cases, the only difference 
was in the classification found, e.g., Mo in one test, S in the other. In 
only five cases was there a discrepancy in toxicity being indicated in one 
test and not in the other. DMSO, Selsun blue and Prell.RTM. showed 
moderate toxicity in the test of the invention, but were non-irritating in 
the Draize tests performed by Applied Biological Sciences. Ajax.RTM. gave 
a non-toxic result in the test of the invention, but was moderately toxic 
in the Draize test. 
The results in Table 2 show that the correlation of the results obtained by 
the method of the invention correlate with human experience approximately 
as well as those obtained from the more laborious, expensive, and 
non-quantitative Draize test. 
In summary, the invention provides a convenient, inexpensive screening 
procedure for obtaining preliminary data with respect to ocular toxicity 
of a material. Results are obtained with comparable reliability to those 
obtained from the relatively non-quantitative non-reproducible and 
expensive procedures, involving whole animals or from alternative, more 
complex, in vitro tests.