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 be absorbed 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 a wider variety of raw materials to achieve the effects he seeks. Furthermore, cosmetic preparations are used far more widely and often than are topical 

drugs, and their use is unsupervised. Thus, unwanted irritant side effects of the vehicle 

are more likely to appear. The attitude of a consumer to any irritation caused by a 

cosmetic is usually much more critical than to a drug product. Therefore, an incidence 

of irritation due to the vehicle of say 1 in 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 doing this, however, we would like to add a word of caution about laboratory 

test methods and their interpretation. These tests need to be run on relatively small 

groups of subjects and then extrapolated to a 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 use conditions, and exaggerate them in a realistic manner. Hopefully, in this 

way, artifacts will not creep into the procedure. Precautions should also be taken to 

choose a test population which has characteristics similar to the intended use population. Certainly, if animal screening tests are employed, the relationship to human 

test procedures needs to be established. 

An exaggerated test protocol having been devised, its validity must be checked with 

actual use experience. That is, the rate of irritant reactions predicted by the laboratory 

method must be compared with the rate observed under normal use conditions. For 

the latter rate, it is fallacious for a manufacturer to assume that it may be calculated 

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. 

74 
METHODS FOR EVALUATING SKIN IRRITANCY 75 

in which the product is placed with a relatively large number of users, and each 

subject is questioned as to irritation side effects as well as product performance. 

The procedures to be described here have been used in our laboratory for a number 

of years and involve tests on human subjects as well 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 the animal skin. It is necessary, therefore, to pretreat 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 area is painted 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 

is applied subsequently. 

The procedure then is as follows. The animal is anesthetized and tied to an 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 1 inch in diameter are marked out on the abdomen. On rats usually 

four such areas may be inscribed, on guinea pigs six areas, and on rabbits 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 each series as a calibration control. 

After all the impregnated pads have been set in contact with the skin, a sheet 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 16 hours the flannel pads are removed, and the sites examined visually. The 

accumulation of blue dye in the various areas can be estimated on a scale 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 in Table 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 TO ANIMALS 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 

100 
76 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 a single application do show a cumulative effect. This has been called 

a “fatigue reaction” in the literature. The degree of reaction is gauged visually from 

the erythema produced. Since we are dealing with human volunteers, a test protocol 

has been devised so that reapplication of the test agent to an already erythematous 

site is avoided. 

To avoid reactions to adhesive tape, special patches were prepared for application 

of the test material to the upper arm. White cotton flannel squares (1.25 x 1.25 inch) 

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 a polyethylene sheet about 2.75 X 2.75 

inches. The flannels are impregnated with the test compositions, and applied to the 

inner surface of the upper arm. These are kept in place 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 17 hours (overnight). The longer duration is used with compositions expected 

to be very low in irritancy. The applications are made daily for four or five consecutive 

days. Readings of erythema are made daily approximately 1 hour after removal of the 

patches, using an eight-point scale as follows: 

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 one grade, 

and only 2.6% differed by more than one grade. 

Whenever a site 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 one of 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 on the arms. 

In order to express the results, a numerical scoring system was devised. This takes 

into account three factors which are multiplied by each other. These are ( 1) score 

for the intensity of the erythema observed, (2) a factor for the number of applications 

necessary to achieve this grade, and (3) a factor to allow for cessation of patching 

early in the test when a strong reaction has occurred. The numerical factors were 
METHODS FOR EVALUATING SKIN IRRITANCY 77 

arbitrarily selected and adjusted so that the irritancy scores obtained with a 

standard irritant when evaluated on 100 subjects would fall into a normal distribution curve with little skewing of the 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” is the 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 

is given in Table 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 a good correlation between the two methods. Another comparison 

is made in Table 4 of a series of shampoo compositions. Here again, a good correlation is seen. However, note the reversal with shampoos 6 and 7. Our feeling is that 

the animal method may be more discriminating. 

TABLE 4 

HUMAN IRRITANCY SCORES AND 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 irritating 
78 P. FINKELSTEIN, K. LADEN, AND W. MIECHOWSKI 

When shampoos 4 and 8 were evaluated for primary irritancy by the procedure 

of Draize et al. (1944), the scores obtained were 2.00 and 2.13, respectively, which 

would classify both formulations as moderately irritating, However, these scoring 

systems would clearly differentiate between them. From use experience it has been 

found that shampoos with scores comparable to shampoo 4 scores sometimes lead to 

some irritancy complaints, but those with scores comparable to shampoo 8 usually 

do not. 

In conclusion then, we believe that these tests are simple to run and lead to 

useful predictions of the irritancy of topically applied preparations. However, they 

are based mainly on observations of changes in capillary dilatation and capillary 

leakage. They may not be well correlated with other kinds of irritancy responses of 

which subjects complain such as stinging or burning. These may require separate 

assessment. 

REFERENCE 

DRAIZE, J. H., WOODARD, G., and CALVERY, H. 0. (1944). Methods for the study of irritation and 

toxicity of substances applies topically to the skin and mucous membranes. J. Pharmacol. I&@. 

Therap. 82, 377-390.