TOXICOLOGY AND APPLIED PHARMACOLOGY 7, 74-78 (1965)



### Laboratory Methods for Evaluating Skin Irritancy P. FINKELSTEIN, R. LADEN, AND W. MIECHOWSKI~ Gillette Medical Research Institute, 6221 North Capital Street, X.E., Washington, D. C. 20011 In the marketing of preparations for use on the skin, a cosmetic manufacturer faces stiffer requirements than does a drug company, at least in certain aspects of product performance. A bland vehicle from which the drug may beabsorbed is the major requirement for the drug company. The cosmetic manufacturer, however, is usually much more concerned with the elegance of the vehicle. Therefore, he needs to employ awider variety of raw materials to achieve the effects heseeks. Furthermore, cosmetic preparations are used far more widely and often than are topical drugs, and their useis unsupervised. Thus, unwanted irritant sideeffects ofthe vehicle are more likely to appear. The attitude of aconsumer to any irritation caused by a cosmetic isusually much more critical than toadrug product. Therefore, an incidence of irritation due to the vehicle of say 1in 10,000 is usually unacceptably high for a cosmetic company. The methods which have usually been used to measure irritancy of preparations for use on the skin are adequate for screening out the stronger irritants but are insensitive in the low irritancy range. We describe below laboratory methods which we have developed and found suitable for this low irritancy range. Before doingthis, however, we would like toadd aword ofcaution about laboratory test methods and their interpretation. These tests need tobe run on relatively small groups of subjects and then extrapolated toa larger and different population. Unless the material under investigation is a strong irritant, exaggerated conditions of exposure to the skin will be required to get an incidence of reaction which may be used as a base for extrapolation. Attempts should be made to simulate, if possible, the intended useconditions, and exaggerate them in arealistic manner. Hopefully, in this way, artifacts will not creep into the procedure. Precautions should also betaken to choose atest population which has characteristics similar to the intended usepopulation. Certainly, if animal screening tests are employed, the relationship to human test procedures needs to beestablished. An exaggerated test protocol having beendevised, its validity must bechecked with actual useexperience. That is,the rate of irritant reactions predicted by the laboratory method must becompared with the rate observed under normal use conditions. For the latter rate, it isfallacious for amanufacturer toassume that it may becalculated simply by dividing the number of irritation complaints received by the total product sales in a given period. Factors such as multiple product purchases by a single individual, lack of reporting of irritation complaints and others can lead to an erroneous estimate. Much more valid is the procedure of conducting consumer tests

1 Deceased.

74METHODS FOR EVALUATING SKIN IRRITANCY 75

in which the product is placed with a relatively large number of users, and each subject isquestioned asto irritation sideeffects aswell asproduct performance. Theprocedures tobedescribed here have beenused inour laboratory for anumber of years and involve tests on human subjects aswell as animals. We have developed confidence in them because they have led to accurate predictions of use experience both in consumer product placement tests and in large scale marketing. With substances of low irritancy, even repeated applications to animal skins give only slight reactions. These are difficult to read because of the normal pinkish color of theanimal skin. It is necessary, therefore, topretreat the application sites to make them more reactive. After examining a number of materials for this purpose, a 20% aqueous formaldehyde solution was chosen. The areaispainted with this solution, and the skin allowed to dry for about 5 minutes. This is repeated three times. The volatility of formaldehyde leaves the skin site uncontaminated when the test material isapplied subsequently. The procedure then isasfollows. The animal isanesthetized and tied toan animal board for the duration of the experiment. Rabbits, rats, and guinea pigs have been employed with equal success. The ventral side of the animal is carefully shaved, and circular areas 1inch indiameter are marked out onthe abdomen. On rats usually four such areas may beinscribed, onguineapigs sixareas, and onrabbits eight. These areas are painted with 20% formaldehyde solution as described above. The test materials are then impregnated on circular white cotton flannel pads 1 inch in diameter and placed over the presensitized test sites. Usually a material of known irritancy is run with eachseries asacalibration control. After all the impregnated pads have been set in contact with the skin, asheet of polyethylene is placed over the entire area. The ends of the sheet are weighted so as to keep the flannel pads in intimate contact with the skin. A 0.5% solution of trypan blue is then injected subcutaneously into the animal at a remote site. After 16hours the flannel pads are removed, and the sites examined visually. The accumulation of bluedye inthevarious areas canbeestimated onascale of 0-lOOy&, or a relative ranking scheme may be employed. We have used both. An example of results obtained by this procedure is shown inTable 1.A series of sodium lauryl sulfate solutions gives the expected increasing irritancy as the concentration is increased. The procedure has been used for emulsion products too, and we shall return to it after first describing our human test procedure.

TABLE 1 EVALUATION OF IRRITANCY TOANIMALS OF A SERIES OF AQUEOUS SOLUTIONS OF SODIUM LAURYL SULFATE
Concentration (%c, Irritancy scorea (%‘o, 0 (water) 1 2 4 6 8

a Most irritating designated 100%. O-5 20 40 60 SO 10076 P. FINKELSTEIN, K. LADEN, AND W. MIECHOWSKI

In this procedure we have tried to exaggerate the irritancy of the materials tested by applying them to the same skin site repeatedly. Many materials that give little response from asingle application do show acumulative effect. This has been called a“fatigue reaction” in the literature. The degree of reaction is gauged visually from the erythema produced. Sincewe are dealing with human volunteers, atest protocol has been devised sothat reapplication of the test agent to an already erythematous site is avoided. To avoid reactions to adhesive tape, special patches were prepared for application ofthe test material tothe upper arm. White cotton flannel squares (1.25 x 1.25inch) were sewn to thin polyethylene backing sheets slightly larger in size. Since it is usually convenient to apply four such patches to each arm, these were usually made up so that four flannel squares were sewn to apolyethylene sheet about 2.75X 2.75 inches. The flannels are impregnated with the test compositions, and applied to the inner surface of the upper arm. These are kept inplace by wrapping the arm with an elastic bandage secured with metal clips. When the patches are removed after each application, the skin site is marked so that another patch can be reapplied to the same site later on. The length of time that the patch is left on the skin is either 5 hours or 17hours (overnight). The longer duration is used with compositions expected tobevery low inirritancy. The applications are madedaily for four orfiveconsecutive days. Readings of erythema are madedaily approximately 1hour after removal of the patches, using aneight-point scaleasfollows:
0 = no visible redness 0 to ? = none to slight redness ? = slight redness ? to k = slight to distinct redness c = distinct redness k to + = distinct to strong reaction
+ = strong reaction f + = very strong reaction We prefer to use designations such as 1 or ? rather than numerals O-7 because the observers seem to keep these in mind better, and are more consistent from day to day. A test of the reproducibility of this scale was made with two trained observers. Out of 1224 readings, 63.1% were in exact agreement, 34.35%differed by onegrade, andonly 2.6% differed by more than onegrade. Whenever asite shows a +- to + or greater reaction, patching at that site is discontinued, although its score is recorded each day. It is found practical to test 15 volunteers simultaneously with eight materials (including oneof known irritancy for calibration purposes). The materials are placed on the arm in a randomized order, and the irritancy readings are made without knowledge of the order in which they were placed onthe arms. In order toexpress the results, anumerical scoring system was devised. This takes into account three factors which are multiplied by each other. These are (1) score for the intensity oftheerythema observed, (2) afactor for the number ofapplications necessary to achieve this grade, and (3) afactor to allow for cessation of patching early in the test when astrong reaction has occurred. The numerical factors wereMETHODS FOR EVALUATING SKIN IRRITANCY 77

arbitrarily selected and adjusted so that the irritancy scores obtained with a standard irritant when evaluated on 100subjects would fall into a normal distribution curve with little skewing ofthe curve. These factors are given in Table 2.

TABLE 2 FACTORS FOR USE IN COMPUTING HUMAN IRRITANCY SCORE+

Intensitv factor Factor for number of Factor for davs of oatchinE cessation of uatchingb 

### ++=so

+ = 40 2+=35 -1- = 30 ??=25 ? 120 0 ? x15 0 =lO 1st day = 2.5 2nd day = 2.0 3rd day = 1.5 4th day = 1.0 5th day = 0.5 1st no patch day = 1.5 2nd no patch day = 2.0 3rd no patch day = 2.5 4th no patch day = 3.0

a The “irritancy score” isthe product of the three factors. b This factor is included only when an area is no longer being patched because of the development of an irritant reaction. Using this procedure, a series of five emulsions was tested. A comparison of the results of the human procedure and the animal procedure for these same materials isgiven inTable 3.
TABLE 3

Emulsion COMPARATIVE IRRITANCY SCORES OF FIVE EMULSIONS Human irritancy
score Animal irritancy score (s)
1 44.5 100 2 34.9 75 3 27.5 45 4 24.3 40 5 21.5 35 These results show agoodcorrelation between thetwo methods. Another comparison is made inTable 4of aseries of shampoo compositions. Here again, agood correlation is seen. However, note the reversal with shampoos 6and 7.Our feeling is that theanimal method may be more discriminating.

TABLE 4 HUMAN IRRITANCY SCORESAND CORRESPONDING DECREE OF IRRITANCY TO

ANIMALS FOR EIGHT SHAMPOOS

Shampoo
1 2 3 4 5 6 7 8 Human irritancy Animal irritancy score rating
46.8 Most irritating 46.0 2nd most irritating 41.8 3rd most irritating 33.3 4th most irritating 27.4 5th most irritating 24.4 6th most irritating 26.7 7th most irritating 24.1 Least irritating78 P. FINKELSTEIN, K. LADEN, AND W. MIECHOWSKI

When shampoos 4 and 8 were evaluated for primary irritancy by the procedure of Draize etal. (1944), thescoresobtainedwere2.00 and 2.13, respectively, which would classify both formulations asmoderately irritating, However, thesescoring systemswouldclearly differentiate betweenthem. From useexperienceit hasbeen foundthat shampoos

with scores

comparabletoshampoo4scoressometimes leadto someirritancy complaints,but thosewith scorescomparableto shampoo8 usually do not. In conclusionthen, we believe that thesetestsare simpleto run and leadto usefulpredictionsof the irritancy of topically applied preparations. However, they are basedmainly onobservationsof changesin capillary dilatation and capillary leakage.They maynot bewellcorrelatedwith otherkindsof irritancy responses of which subjectscomplainsuchasstinging or burning. Thesemay require separate assessment. REFERENCE DRAIZE, J. H., WOODARD, G.,and CALVERY, H. 0. (1944). Methods for the study ofirritation and toxicity ofsubstancesapplies topically to the skin and mucous membranes. J. Pharmacol. I&@. Therap. 82, 377-390.