TOXICOLOGY AND APPLIED PHARMACOLOGY 7, 308-319 (1965)



### Comparative Evaluation and the Influence of Various

Factors on Eye-Irritation Scores
A. S.WELTMAN,S. B. SPARBER,AND TANIA JURTSHUK~ Brooklyn College of Pharmacy, Long Island University, Brooklyn, New York, and

St. Vincent Hospital, New York, New York

Received April 13, 1964 Safety-testing measures requireanddemandtheeliminationofunsafe,harmful, and deleterioussubstancesfrom product developmentprior to their releasefor public consumption.Unfortunately, the developmentof new,and perhapsbeneficial,productshasbeenblockedtemporarily orpermanentlyby “pass-fail” type of examinations basedonconclusionsdrawnfromsomewhatlimited studies. The scoringprocedureoutlined by Draize etal. (1944) andits subsequentmodification by Draize (1959) have beenthe methodsmostfrequently utilized for the interpretation andevaluation of eye-irritation tests.Ophthalmic medicationsor cosmetic products, suchasshampoos,likely to comein contact with the humaneye, mustof necessitypassthis type or related kindsof examinations.Although, through theyears, the Draize techniquehasperformed anadmirable function in the testing andpreventative marketing of harmful substances,certain shortcomingsinherent in theprocedurehavebecomeevident. Recently, RussellandHoch (1962) reportedfive somewhatdiversecomparativeevaluationsmadeindependentlyby five testinglaboratoriesoftwoshampoo

samples scoredby theDraize test.Sinceeachofthelaboratories representedwell-known and reputable biological testing centers,the questionarises asto the possiblecauseor causesof thesedeviations and how suchdifferencesin scoringcanbeeliminatedor explained. The Draize technique, which utilizes the albino rabbit, subjectively and quantitatively scoreseyeirritation by observingcorneal, iridial, and conjunctival damage following instillation of O.l-ml amountsof the test material in oneeye, the other untreatedeyeservingasacontrol. A series of 9rabbitsareusuallyusedfor eachtest. The treated eyesof 3rabbits remainunwashed.In contrast, to observepossibleprevention of injury, thetreated eyesof 3other rabbitsarewashedwith 20mlof lukewarmwater 2seconds

after instillation andtheremaining3 rabbitsat 4seconds after instillation of thetestmaterial.Ocular readingsaremadeat 24,48,and72hoursand at 4and7daysafter treatment oraslongasthe injury persists. The actual grading processissubjective and involved; this in itself representsa largesourcefor the disparities in theconclusionsof various individuals scoringthe reactions.Quantitatively, cornea&iridial, andconjunctival damageare not evaluated equally. Cornea1damage,by far, possesses thegreatestweighting factor basedupon considerationsof its importancetovisionanditsgreatersusceptibility todamage. Fully realizing the extreme difficulty in adequately devisinga numerical scoring

1 Senior resident ophthalmologist, St. Vincent Hospital.EVALUATION OF EYE-IRRITATION SCORES 309

system for classifying eye damage, the investigation sought to combine with the Draize procedure, the simultaneous use of additional techniques involving photographic, ophthalmoscopic and histologic observations. It was hoped that procedures such as these would aid in the reduction of subjective errors and help in the proper evaluation and assessment of eye irritation and the substances capable of inducing such damage. The study also considers the role of population sizes, time of release, and other factors which may affect eye irritation scores,

METHODS The shampoo preparations studied were supplied by the Malmstrom Chemical Corporation and were identified by the numbers A-65/1 and A-65/2. They were from the same batches submitted by Russell and Hoch (1962) to five commercial laboratories for testing by the Draize method. Their compositions are shown in Table 1.

TABLE 1 COMPOSTION OF SHAMPOO PREPARATION TESTED Shampoo preparation A-65/1 A-65/2 (%) (%)
TEA lauryl sulfated (40% solution) 50.0 50.0 Deionized water 41.0 45.0 Lanolin alcohol@ with 16moles C,H,O 3.0 - Lanolin oil* 1.0 - CCOFA diethanolamidec 5.0 5.0
100.0 100.0

a Nimcolan@ “S’‘-Malmstrom Chemical Corporation. b Lantrol@-Malmstrom Chemical Corporation. 0 Sandrol superamide lOOC-Clintwood Chemical Company. d Duponol wat.
The reactions were scored according to the Draize scale with certain modifications at the l-, 2-, 3-, 4, and 7-day intervals. In addition, eye damage was also scored
on the 5th day. The modifications consisted mainly in recording changes such as lack of luster, in noting intermediate grades or effects (i.e., l+ or 2+, etc.), and in attempting to determine the range and degree of subjective errors introduced by borderline evaluations on the total mean scores. Too often borderline alterations create personal and subjective difficulties in assigning avalue to that change (i.e., whether to score such event with ahigher or lower unit value). To assay this factor, borderline cases were recorded as minus numbers. The data were then calculated on the basis of high-scoring and low-scorin gestimates. For example, the borderline grade of minus 2was computed as either having anumber 2or number 1 Draize value when mean scores were calculated. Twenty-eight young male albino rabbits averaging 1.96 kg were used. The rabbits were acclimated and housed for 1week prior to initiation of the study in alaboratory maintained at 70°F. In general, a modified Draize technique was followed which omitted the eye-washing stages to attain maximum damage. Twenty-four of the rabbits were used as test animals; they were divided into groups of four. The procedure entailed the instillation of O.l-ml amounts of the respective test solution into the310 A. S. WELTMAN, S. B. SPARBER, AND TANYA JURTSHUK

conjunctival sac of each eye by means of a tuberculin syringe. The eyes of all test and control rabbits were examined for gross defects before instillation of the test or saline solutions. To prevent loss or escape of the fluid from the sac, the eyelids were held in a closed position away from the eyeball for approximately 20 seconds. The opposite eye was used to compare the effects of the second sample in order that the effects of both solutions could be evaluated on the same individual. To avoid scoring errors due to possible differential reactivities and sensitivities of either the right or left eyes, each test preparation was instilled into 2left and 2right eyes of each group of 4 rabbits. The reactions were scored according to the Draize scale at intervals of 1, 2,3, 4, and 7days. In addition, eye damage was also scored on the 5th day. The knowledge to score eye damage was acquired by prior visits to two testing laboratories and by consultation with the Food and Drug Administration, Washington, D. C. The lighting consisted of overhead fluorescent lights assisted by daylight from an adjacent and nearby window. The extent of eye damage was read with the unaided eye and with an ophthalmoscope. Four rabbits were used as control animals. Identical volumes of normal saline were administered into 2 left and 2 right eyes of the control group, the corresponding opposite eyes remaining untreated. All saline-treated and untreated eyes were examined and scored in asimilar fashion asthe test eyes. Four test rabbits were sacrificed daily at days 1, 2, 3, 5, and 7by sodium pentobarbital injections administered into the ear veins of the respective rabbits. Immediately prior to autopsy, however, two photographs were taken with a Minox III B camera of each eye, using Kodachrome film (ASA-25). One picture was taken of the treated eye at rest. The second picture attempted to demonstrate the extent of corneal, iridial, and conjunctival damage by holding apart the upper and lower eyelids. The choice of the Minix III B camera was twofold: (a) its simplicity of operation without the need of additional devices such as close-up lenses and (b) the ease by which all 4 photographs of the same animal could be mounted on a conventional 35mm frame for simultaneous projection and evaluation. Upon autopsy, both eyes of each animal were surgically removed, labeled, and fixed in Bouin’s solution. The tissues were embedded in Bioloid paraffin (56-SS”C), sectioned at 4 p, and stained with hematoxylin and eosin. Histologic observations were based on three slides derived from three regions of each eye. Vertical cross sections were prepared of the mid-region as well as extreme right and left areas of each eye. Every slide contained two adjacent sections taken from one of the respective areas being studied. All twenty test rabbits sacrificed on days 1, 2,3, 5, and 7for histologic purposes were released immediately following instillation of the test preparations. An additional group of 4rabbits were held in stocks and released 1% hours after instillation of the test samples. Although the eyes of these rabbits were not prepared histologically, the animals were graded and examined in asimilar fashion asthe others for 7days.

OBSERVATIONS A. Extent of Personal and Subjective Errors on the Mean, Total Eye-l&at& Scores
Table 2presents thehigh- and low-scoring estimates of the mean total eye-irritation grades recorded by visual examination of the rabbits’ eyes. These estimates reveal theEVALUATION OF EYE-IRRITATION SCORES 311

range of subjective error and borderline doubts onthe mean, total eye-irritation scores. The data were compiled from an initial population of 24 test rabbits subsequently reduced to asample size of 8rabbits by the gradual autopsy procedures. Analyses demonstrated noappreciable or statistically significant differences between the effects produced by samples A-65/1 and A-65/2 whether computed on a high- or low-scoring basis. Furthermore, no significant differences were observed between the respective high-score or low-score estimates of each test solution. This serves to demonstrate the range of the differences which anindividual’s subjective error can produce ininitial scoring procedures. In this instance, the differences were relatively minimal.

TABLE 2 MEAN SCORES OF ALBINO MALE RABBIT EYE-IRRITATION TESTS: RESULTS COMPARE

HIGH-SCORE AND LOW-SCORE ESTIMATES

Parameter Number of rabbits High-score estimate SampleA-65/1 SampleA-65/2 P value (A-65/1 vs. A-65/2) Low-score estimate SampleA-65/1 SampleA-65/2 P value (A-65/1 vs. A-65/2) P valuesof differencesbetween high- and low-score estimates A-65/1 (high-score vs. low-score) A-65/2 (high-score vs. low-score) 1st 2nd

24 20

16.5 15.9 17.4 15.1 0.81 0.86

13.5 13.3 12.1 10.5 0.66 0.49

0.41 0.58 0.11 0.22 Day scored

3rd 4th 5th 7th
16 12 12 8

9.8 5.3 0.8 0.0 9.9 3.5 0.2 0.0 0.90 0.61 0.23 -

8.4 4.5 0.0 0.0 9.2 2.0 0.0 0.0 0.85 0.48 - -

0.74 0.85 - - 0.87 0.55 - -
B. Estimate of Sample Sizeas a Factor Ajecting Mean Total Score As stated previously, the Draize procedure requires the testing of a minimum of 9 rabbits observed for a total of 7 days, or longer if required. Table 3presents the
mean total scores compiled from 8rabbits observed throughout the 7-day period. To avoid bias and to simplify calculations, in the following comparisons only the highscore estimates are tabulated. Statistical analyses of the data derived from the population of 8 rabbits again revealed no significant differences between the eye-irritation scores produced by the two samples.

TABLE 3

MEAN SCORES

OF ALBINO MALE RABBIT EYE-IRRITATION TESTS: RESULTS COMPARE HIGH-SCORE ESTIMATES BASED ON A POPULATION OF EIGHT RABBITS Day scored Parameter 1st 2nd 3rd 4th 5th 7th
Number of rabbits 8 8 8 8 8 8 High-score estimate Sample A-65/1 17.9 18.4 6.8 4.5 0.8 0.0 Sample A-65/2 20.9 19.4 7.3 1.8 0.0 0.0 P value (A-65/1 vs. A-65/2) 0.70 0.90 0.90 0.42 - -312 A. S. WELTMAN, S. B. SPARBER, AND TANYA JURTSHUK

Table 4 reviews and compares the mean total scores obtained from the complete population of 24test rabbits, apopulation of 8test rabbits and the 2subgroup populations of 4 rabbits each. It should be noted that while one group of 4 rabbits was released immediately (in accord with the Draize procedure) the other group of 4 rabbits was kept in stocks for a period of lyZ hours after instillation of the test substances. It is apparent that the differences and ranges of the various mean total
scores showed a greater degree of variability and spread as the population sizes decreased. This was especially illustrated in the range of differences displayed by the

TABLE 4 SCORES OF ALBINO MALE RABBIT EYE-IRRITATION TESTS: RESULTS COMPARE SCORES DERIVED FROM POPULATIONS OF VARYING SAMPLE SIZES Day scored
G~OUD n” 1st 2nd 3rd 4th 5th 7th High-score estimate A-65/1 (complete pop.) A-65/1 A-65/1 (release delayed) A-65/1 (released immed.) A-65/2 (complete pop.) A-65/2 A-65/2 (release delayed) A-65/2 (released immed.) P value (A-65/1 release delayed
vs. released immed.) P value (A-65/2 release delayed
vs. released immed.) * Number of rabbits. 24-8 16.5 15.9 9.8 5.3 0.8 0.0 8 17.9 18.4 6.8 4.5 0.8 0.0 4 29.3 31.3 12.5 9.0 1.5 0.0 4 6.5 5.5 1.0 0.0 0.0 0.0 24-S 17.4 15.1 9.9 3.5 0.2 0.0 8 20.9 19.4 7.3 1.8 0.0 0.0 4 21.8 20.8 5.5 2.5 0.0 0.0 4 20.0 18.0 9.0 1.o 0.0 0.0

0.02 0.02 0.14 - - -

0.88 0.80 0.64 0.32 - -

A-65/1 data. Statistical analyses of the mean total scores of the 2 subgroup populations of 4 rabbits indicated significant differences between the scores of the A-6.5/1 series on the 1st and 2nd days (i.e., 29.3vs. 6.5and 31.3 vs. 5.5). No significant differences were observed between the mean total of the A-65/2 sample also based on apopulation of 4 rabbits (i.e., 21.8 vs. 20.0 and 20.8 vs. 18.0). The variability due to population sizes was less apparent in that series. The differences, however, between the values of the 2subgroup populations of 4 rabbits causes one to reflect on timerelease factors affecting eye-score evaluations.
C. Estimate of Time of ReleaseasaFactor Afecting Mean Total Scores A question requiring resolution iswhether the vigorous eye-rubbing and pawing activities ofatreatedrabbit canaggravateandcauseunduedamageaboveandbeyond that inducedby thetestsubstances. In oneinstance,apuncture lacerationwasnoted onthe uppereyelid of atest rabbit which hadbeenreleasedimmediately. Table 5presentsthe effectsof immediateversusdelayed releaseof the rabbits as a factor influencing eye-irritation scores.The valueswereobtainedby totaling the combinedscoresof 8eyesof the 4 rabbitstreated and released immediately ascomparedto the scoresobtained from the 8eyesof 4 rabbits immobilized in stocksfor 1% hours.To increasesamplesizealiberty wastaken in combiningtheA-6.5/1 andEVALUATION OF EYE-IRRITATION SCORES 313

A-65/2 data in view of the absence of any statistically significant differences between the alterations produced by the 2 solutions on the basis of the complete population analyses. The data indicated that, in general, a lesser degree of eye irritation was observed in the rabbits released immediately versus those immobilized by the stocks. In some instances, these differences approached statistical levels of significance as shown by the P values on the lst, 2nd, and 4th days. It should be recalled that although significant differences were observed in the A-65/1 series on the 1st and 2nd days (Table 4, n=4) none were noted in the comparable A-65/2 group. Further
TABLE 5 SCORES OF ALBINO MALE RABBIT EYE-IRRITATION TESTS: RESULTS COMPARE

EFFECTS OF DELAYED SCORES vs. IMMEDIATE RELEASE Day scored Group ian 1st 2nd 3rd 4th 5th High-score estimate Delayed release 8 25.5 26.0 9.0 5.8 0.8 Immediate release 8 13.3 11.8 5.0 0.5 0.0 P value (delayed vs.immed. release) 0.10 0.06 0.43 0.10 - a Number ofobservations: 8eyesof4 rabbits. 7th

0.0 0.0

studiesare therefore required to determinewhether thesefindings representchance occurrencedueto smallpopulation sizesor a demonstrationof the beneficialeffects whicheye-rubbing may causeby stimulating thelacrimating glandsecretionstowash thetreatedeyes.Thiswouldseeminglycounteractthedangerof injuriesorirritations causedby theclumsy, pawingactivities of the rabbits immediately following instillation of asampleto therabbits’eyes. In general, histologic examination between control andthevariously treated eyes revealednosubstantialdifferencesbetweenthe effectsof either of thetwo testsubstanceson the microscopicstructure of the treated eyes.Moreover, a finedegreeof correlation andcorrespondence wasnotedbetweentheeye-irritation scores determined at thetimeofsacrificeandthedegreeofhistologicdamage.Primarily, alltheobserved histologicalterations werelimited to the cornea,specifically the stratified squamous epitheliumandthe substantiapropria layers. No apparent involvementswerefound either in the iris or in the choroid and retinal layers. The stratified epithelial layersof the normal rabbit corneaconsistsof onelayer of columnar cells located beneath 3 to 5 rows of nonkeratinizing polyhedral and squamous

cells.Posteriorly, onefindsthe Bowman’smembranefollowedby the relatively thick substantiapropriacomposed of modifiedconnectivetissue,theDescemet’s membrane,and lastly asinglelayer of endothelialcells.The epithelium layer of the bulbar conjunctiva mergesindistinguishably with that of the corneaat the region of thelimbus(Prince etal., 1960). Although approximately one-third of the eyestreated by either of the two substancesappearedhistologically normal,approximately two-thirds of the treated eyes showedconsiderableerosioninthestratified squamous layersof thecorneaduring the first 3daysfollowing instillation of thetestpreparations.Thedamagedeyesrevealed little or nostratified squamous epithelial cells.Accompanying this epithelial erosion,314 A. S. WELTMAN, S. B. SPARBER, AND TANYA JURTSHUK

one found substantial increases in leucocytic and lymphoid cellular infiltrations of the substantia propria. The progressive regenerative and repairing capacities of the cornea were demonstrated however by the microscopic observations which revealed that by the 5th day only one of the treated eyes showed remnants of slight damage to the stratified squamous epithelium. By the 7th day, all the eyes treated by both substances were histologically normal in appearance.

The photographic plates (Figs. 1-S) illustrate the extent of the structural changes, epithelial erosion, and leucocytic infiltration caused by addition of either of the 2 samples to the eyes of rabbits. The plates further illustrate the regenerative repair of the epithelial layers by the 5th and 7th days as compared to the normal saline or untreated eyes.

Examination of the transparencies demonstrated likewise that the external, physical appearance of the test and control eyes corresponded to the condition shown by the various histologic plates. The test solutions primarily produced alterations in the

FIG. 1. Longitudinal section of cornea of rabbit sacrificed at the 24-hour period after treatment with solution A-65/1. Section illustrates erosion and loss of stratified epithelial layers with dense leucocytic infiltration in the substantia propria. Hematoxylin and eosin. Magnification: X 400.

FIG. 2. Low power magnification of longitudinal section of cornea of rabbit sacrificed 3 days after treatment with solution A-65/2. Section demonstrates partial regenerative repair of epithelial layers with persistent dense leucocytic infiltration in the substantia propria. Hematoxylin and eosin. Magnification: X 100.315

FIG. 3. High power (X 400) magnification of Fig. 2.

FIG. A-65/1 dence
x 400. 4. Longitudinal section of cornea of rabbit sacrificed 5 days after treatment with solution

. Section illustrates relatively normal appearance of epithelial layers with some slight eviof leucocytic infiltration in the substantia propria. Hematoxylin and eosin. Magnification:

FIG. 5. Low power magnification of longitudinal section of cornea of rabbit sacrificed 7 days after treatment with solution A-65/1. Section demonstrates normal appearance of epithelial layers and th ,e absence of leucocytic infiltration in the substantia propria. Hematoxylin and eosin. Magnificati on: X 100.316 A. S. WELTMAN, S. B. SPARBER, AND TANYA JURTSHUK

FIG. 6. High power (X 400) magnification of Fig. 5.

FIG. 7. Lsow power magnification of longitudinal section of cornea of sali demor istra rtir4 normal appearance of epithelial layers and substantia propria. eosin. Ma gnificartion: X 100. neh-treated con!.rol H[ematox] 7lin a .nd

FIG. 8. High power (X 400) magnification of Fig. 7.EVALUATION OF EYE-IRRITATION SCORES 317

conjunctiva and discharges from the eyes of treated rabbits. By the 5th and 7th days no permanent damage was observed in any of the corneas and eye discharge appeared normal. Analyses of the ophthalmoscopic observations revealed that the two test samples had little or no effect on the internal structure of the rabbits’ eyes. The fundi, as well asthe crystalline lenses, were grossly normal in appearance and seemed unaffected by the test compounds. However, in some instances, there was a certain degree of difficulty in obtaining clear visualization of the fundus of the retina. The degree of difficulty corresponded with and showed a positive correlation with the total mean and cornea1 scores of the various test animals in much the same fashion as did the histologic observations.
DISCUSSION It is evident from the data that both substances compare favorably and almost equally alike with regard to their eye-irritation properties. The Draize test, the ophthalmoscopic, photographic, and histologic observations revealed that the extent or lack of damage caused by either of the two samples ranged within the same general category. Recently, Kay and Calandra (1962) have presented amethod for the interpretation of eye-irritation scores based on the Draize technique. Use of their empirical method aided in the evaluation of both samples as preparations possessing mildly irritating properties. However, the data do demonstrate or suggest that personal error, sample size, and time of release can or may affect total mean scores. Although personal scoring or subjective errors were found to have a relatively minor influence (the present scores being based on the simultaneous evaluations and agreement by three individuals), this study cannot rule out the possibility that the variations between the scores of differently trained individuals from two laboratories may be considerable. Such instances would serve to emphasize the necessity of additional detailed standardization of the Draize technique to permit greater uniformity of individual evaluation and assessment of eye damage. The study similarly demonstrates that depending on sample sizes one can have considerable differences between the total mean scores of various test groups (i.e., groups of 4 vs. 8 vs. 24). Statistical analyses have amply demonstrated the greater validity of values based on samples of sufficient population sizes. Further enlargement of the number of rabbits used in the standard Draize method would be helpful in avoiding the “pass-fail” type of errors possible by chance occurrence when small populations are employed. Concerning the factor of time of release, additional investigations are required to determine the beneficial or deleterious role which either early or late release may have on eye-irritation scores. Cursory examination always showed that within minutes following release (delayed or immediate) the regions and hair surrounding the eyes acquired a wet and matted appearance and the eyes appeared more inflamed and redder. Whether early release aids in the washing out or the dilution of the substances or whether the vigorous, unrestrained and clumsy pawing activity in the vicinity of the eyes can aggravate damage requires further analysis. The present evidence would indicate that on the whole, early release had ultimately a beneficial effect, perhaps318 A. S. WELTMAN, S. B. SPARBER, AND TANYA JURTSHUK

attributable to eye-washing, although the sample size in the present study was small. If so, the habits of different laboratories concerning the time of release of the test animals could have considerable influence on total mean scores aswell as between the scores of different laboratories. Possibly the greatest variable has been relatively neglected, that of the animal itself. Unfortunately, rabbit strains donot have theuniformity and standardization of strains of other species of laboratory animals, and unfortunately the breeding facilities of various farms supplying rabbits are far from satisfactory. Age, sex, and strain differences can affect the resistance of laboratory animals to either chemical or physical states of stress. To illustrate, certain strains of mice and rats are more prone to audiogenic seizures (Hamburgh and Vicari, 1960; Finger, 1947). Similarly, differences in seizure-susceptibility frequencies have been reported both on an age (Ginsburg and Hovda, 1947; Maier and Glaser, 1942), and sex (Ginsburg and Hovda, 1947; Finger, 1943) basis in mice and rats. Probably, in many instances, the difficulties in obtaining healthy rabbits from acceptable breeding farms might cause testing laboratories to overlook or underate sex, age,and strain difference factors. Yet, influences such asthese may well bethe prime
reason for the diverse scores of different testing laboratories, as for example, those reported by Russell and Hoch (1962). A serious deficiency arises, therefore, in that the Draize procedure does not necessarily stipulate age,sex, or strain requirements other than that the animals be albino rabbits. Here, again additional standardization is required. A truly severe and critical procedure would entail the testing of animals belonging to the more susceptible sex and at their most susceptible agelevel. In attempting to interpret the usefulness of photographic, ophthalmoscopic and histologic methods in assessing eye-irritation damage, the mildly irritating nature of the present two test samples limited the observable severity of eye damage. However, the techniques can beinvaluable adjuncts to standard Draize test procedures: 1. The photographic method can permanently record the external appearance and physical state of the eyeat any period within certain limits. 2. Ophthalmoscopic techniques can aid in prompt visualization of retinal damage. A
more precise and invaluable tool is the slit-lamp ophthalmoscope. This instrument determines not only the location, extent, and depth of corneal, iridial, lens, and retinal damages, but also changes in the anterior and posterior chambers of the eyes. 3. Histologic procedures can fully ascertain and permanently record the structural damage done to the tissues, layers, and cells of the eyes. The authors recognize that these additional techniques could contribute materially toward increasing the cost of proving and demonstrating the value of a substance. On the other hand, the techniques could materially aid in granting a truer perspective of the value of anew product that ordinarily might have been withdrawn from development solely on the basis of premature conclusions on eye irritation based on insufficient tests. The importance of fully determining the damaging potentialities of any new product must not beunderestimated.EVALUATION OF EYE-IRRITATION SCORES 319

SUMMARY
This study employed ophthalmoscopic, photographic, and histologic techniques as an adjunct to standard Draize procedures in interpreting and evahrating the degree of eye irritation produced by two solutions on the cornea, iris, and conjunctivae of rabbits’ eyes. Although ophthalmoscopic examinations revealed no evidence of retinal or internal damage, histologic observations, in some instances, demonstrated considerable erosion and loss of the stratified epithelial layers with evidence of dense leucocytic infiltration in the substantia propria of the cornea. By the 5th and 7th days, however, all treated eyes appeared histologically normal. The various data indicated that there were no significant differences in the degree of eye irritation caused by either preparation and that the solutions possessed properties of a mildly irritating nature. Certain inadequacies in obtaining a correct evaluation of eye irritation by the Draize technique were noted. In particular, the influence of such factors as subjective and personal error, sample size, time of release as well as age, sex, and strain differences of rabbits were discussed. It is believed that further standardization and modification of the Draize procedure is required to eliminate discrepancies between the scores of individual testing laboratories as well as differences between the mean scores of two or more runs of the same preparation.

REFERENCES DRAIZE, J. H. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. of Food and Drug Officials of the U.S., pp. 49-52. Editorial Committee, Baltimore, Maryland. DRAIZE, J. H., WOODARD, G., and CALVERY, H. 0. (1944). Methods for the study of irritation and toxicity of substances applied topically to the shin and mucous membranes. /. Pharm. Exptl. Therap. 92, 377. FINGER, F. W. (1943). Factors influencing audiogenic seizures in the rat. J. Camp. Psychol. 36, 227-232. FINGER, F. W. (1947). Convulsive behavior in the rat. Psychol. Bull. 44, 201-248. GINSBURG, B. E., and HOVDA, R. B. (1947). On the physiology of gene controlled audiogenic seizures in mice. Anat. Record 99, 621. HAMBURG=, M., and VICARI, E. (1960). A study of some physiological mechanisms underlying susceptibility to audiogenic seizures in mice. J. Neuropathol. Exptl. Neurol. 19, 461-472. KAY, J. H., and CALANDRA, J. C. (1962). Interpretation of eye irritation tests. J. Sot. Cosmetic Chemists 13, 281. MAIER, N. R. F., and GLASER, N. M. (1942). Studies of abnormal behavior in the rat. J. Comp. Psychol. 34, 23-28. PRINCE, J. H., DIESEM, C. D., EGLITIS, I., and RUSKELL, G. L. (1960). Anatomy and Histology of the Eye and Orbit in Domestic Animals. Thomas, Springfield, Illinois. RUSSELL, K. L., and HOCH, S. G. (1962). Product development and rabbit eye irritation. Proc, Sci. Sect. Toilet Goods Assoc. 37, 27.