Optimal patch application time in the evaluation of skin irritation

Authors


Correspondence: Hiromi Kanto, M.D., Ph.D. First Department of Dermatology, Toho University School of Medicine, 6-11-1 Omorinishi, Otaku, Tokyo, 143-8541, Japan. Email: derma1st@med.toho-u.ac.jp

Abstract

We investigated the optimum application for evaluating skin irritation response by using samples of irritants commonly used as additives in cosmetics and other common household products. We studied 47 volunteers (16 men and 31 women). We selected three types of surfactant, one moisturizer, one anti-infective agent and one oil solution. Using Finn chambers on Scanpor tape, we performed the patch test. A total of 0.015 mL of each sample was applied to the Finn chamber. For liquids, circular filter paper was soaked in 0.015 mL of the sample. Samples were placed on the upper back of participants, and closed for 4, 24 or 48 h. A patch application time of 24 h is sufficient to detect primary skin irritation from irritants in cosmetics and other common household products. In addition, we found that skin irritation reactions were strongest at 24 h after patch removal and that the reaction tended to be weaker at 48 h after patch removal. Patch testing to evaluate irritants should be performed by means of a 24-h patch test with a follow-up reading at 24 h after patch removal. An application time of 24 h places less of a burden on patients than a 48-h patch test.

Introduction

Human patch tests are often used to identify the substances (allergens) responsible for contact dermatitis.[1, 2] Samples of various substances that may cause contact dermatitis are applied to a healthy area of the skin under a patch. They remain on for a period of time, usually 48 h, after which they are removed. Readings are taken at 1–2 h after and at 24 h after removal of the samples. An additional reading may be taken at 1 week to 10 days after application of the patch, not depending on the strength of the reactions at 72 h exhibited by the patient.

In standard clinical practice, chemicals and cosmetics are applied to the skin on a patch to determine responses to irritation, as well as allergic reactions. Medical institutions use an identical testing procedure to evaluate response to irritation and allergic response; there is no special patch test used only for evaluating skin irritation. There have been reports outside Japan of sodium lauryl sulfate (SLS) being used to evaluate skin irritation.[3, 4] SLS has been used in closed patch tests to evaluate skin irritation, with application times of either 23–24 h[5-8] or 4 h.[9-14] In particular, closed patch tests with an application time of 4 h have been used to detect comparatively strong skin irritations, such as those involving skin irritants and substances corrosive to the skin in relation to regulations involving chemical substances.[9, 10]

In the present study, we investigated the optimum application time for evaluating skin irritation response by using samples of irritants commonly used as additives in cosmetics and other common household products. The substances are known to cause an irritation response in people, although they are not strong irritants.

Methods

Participants

We studied 47 volunteers (16 men and 31 women), who all gave informed consent before participating in the study. The age of participants ranged 23–61 years, with a mean of 37.1 years.

The inclusion criteria were: healthy back skin (i.e. no dermatological conditions, inflammation, or desquamation caused by dryness), no oral or topical steroid use and no known allergies to patch test samples.

Patch test materials

Table 1 shows the samples used in the study. We selected three types of surfactant, one moisturizer, one anti-infective agent and one oil solution. It has been shown that although these substances are not strong irritants, they are suitable for observing irritation reactions in humans and animals. The concentration of samples was adjusted to a level that would likely cause irritation. Distilled water was used in the study as a solvent control, and white petrolatum and saline solution were used as negative controls. The identity of patch test samples was not revealed until the study was completed.

Table 1. Patch test materials
NoSampleConcentration (%)CAS No
  1. CAS: Chemical Abstracts Service.

1Sodium lauryl sulfate (SLS)0.5151-21-3
2 0.3 
3 0.1 
4Propylene glycol (PG)50.057-55-6
5 30.0 
6Sodium laurate (SL)2.0629-25-4
7 1.0 
8Isopropyl myristate (IPM)100.0110-27-0
9Benzalkonium chloride (BC)0.18001-54-5
10 0.05 
11PolyOxietylene (lO) oleyl ether 10.08009-03-8(NF)
12(Oleth-10)5.0 
13White petrolatum (WP) solventas is8009-03-8(NF)
14Distilled water (DW) solventas is 
15Saline negative controlas is 

Patch test

Finn chambers (Epitest Ltd Oy., Tuusula, Finland) (five chambers per strip) on Scanpor tape (Alpharma AS, Norway) were used for the patch test. The size of Finn chamber was 11 mm and aluminum cup was 8 mm. A total of 0.015 mL of each sample was applied to the Finn chamber. For liquids, circular filter paper was soaked in 0.015 mL of the sample. Samples were placed on the upper back of participants, and closed for 4, 24 or 48 h (Fig. 1). Surgical tape was used as necessary to secure patch test units and to prevent peeling of adjacent skin.

Figure 1.

The test schedule.

Assessment of participants

Participants were assessed by dermatologists at the Department of Dermatology, Toho University Omori Medical Center; Department of Dermatology, Hyogo College of Medicine; and Department of Dermatology, Fujita Health University School of Medicine. Follow-up readings were performed 2, 24 and 48 h after patch removal. Skin response was evaluated according to two sets of criteria: the new criteria for skin irritation of the Skin Irritation Research Group of the Japanese Society for Contact Dermatitis[15, 16] and the standard Japanese criteria (Table 2).[2]

Table 2. The new criteria for skin irritation of the skin irritation research group of the Japanese society for contact dermatitis
New standard for evaluating skin irritationaScoreJapanese StandardbSugai Scoringc
  1. a

    Kawai15.

  2. b

    Kawamura2.

  3. c

    Sugai20.

No reaction00
Perceptible erythema1– or±0 or 0.5
Weak-to-moderate erythema2±0.5
Distinct erythema3+1
Erythema with popular or edematous reaction4++2
Erythema with vesicular reaction5+++3
Corrosive reaction (bullae formationnecrosis)6++++4

Approval was obtained from the Ethics Committee of the Japanese Society for Dermatoallergology and Contact Dermatitis before commencing the study.

Statistical analysis

The χ2-test was used in the comparison of the incidence of the new criteria for skin irritation and the standard Japanese criteria after 4, 24 and 48 h. P < 0.05 was considered to be significant.

Results

Figure 2 and Table 3 show the results of the patch test at different application times, according to the new criteria for skin irritation Similarly, Figure 3 and Table 4 show the results of the patch test at different application times, according to the standard Japanese criteria. The incidences of skin reactions after 4 h of application were the lowest of all three follow-up time points. This was true using both sets of evaluation criteria. For application times of 24 and 48 h, the incidence of skin reactions was usually higher at a follow-up time of 24 h after patch removal. After 4 h of application, follow-up time was not associated with the incidence of skin reactions.

Table 3. The summary of results in new standard
ChemSampleConc. (%)hourEvaluation 2 h afterEvaluation 24 h afterEvaluation 48 h after
MeanSDMaxMinMeanSDMaxMinMeanSDMaxMin
C01SLS0.548 h1.26 1.09 301.98 1.22 401.43 1.08 30
C01241.36 1.22 401.94 1.24 401.19 1.14 40
C0140.15 0.36 100.32 0.73 300.19 0.54 20
C02SLS0.348 h0.85 0.91 301.74 1.33 401.11 1.13 30
C02240.60 0.88 301.28 1.23 400.74 1.01 30
C0240.13 0.40 200.21 0.55 200.11 0.31 10
C03SLS0.148 h0.23 0.52 200.55 0.83 300.30 0.59 30
C03240.32 0.66 300.60 0.88 300.32 0.59 20
C0340.23 0.52 200.00 0.00 000.06 0.32 20
C04PG5048 h1.00 0.91 300.64 0.82 300.40 0.65 20
C04240.66 1.03 400.28 0.50 200.11 0.31 10
C0440.04 0.20 100.06 0.25 100.11 0.31 10
C05PG3048 h0.85 0.96 400.68 0.78 200.45 0.75 30
C05240.38 0.68 200.26 0.53 200.21 0.46 20
C0540.02 0.15 100.00 0.00 000.09 0.28 10
C06SL248 h1.30 1.08 401.13 1.03 300.70 0.91 30
C06241.40 1.08 301.38 1.07 301.11 1.03 30
C0641.00 1.23 400.49 0.75 200.23 0.63 30
C07SL148 h0.55 0.77 300.34 0.56 200.19 0.50 20
C07240.66 0.76 300.64 0.70 200.38 0.61 20
C0740.53 0.93 300.19 0.45 200.15 0.47 20
C08IPM10048 h0.23 0.52 200.11 0.31 100.21 0.46 20
C08240.04 0.20 100.04 0.20 100.15 0.55 30
C0840.04 0.20 100.00 0.00 000.13 0.45 20
C09BC0.148 h1.09 1.08 300.79 0.88 300.72 0.99 30
C09241.11 1.34 400.87 1.10 400.55 0.83 30
C0940.32 0.59 200.02 0.15 100.04 0.20 10
C10BC0.0548 h0.77 0.94 300.55 0.77 300.45 0.69 30
C10240.40 0.65 200.30 0.69 400.21 0.59 30
C1040.13 0.45 200.09 0.28 100.04 0.20 10
C11Oleth-101048 h0.49 0.78 200.43 0.62 200.17 0.48 20
C11240.38 0.80 300.17 0.52 200.26 0.49 20
C1140.13 0.40 200.04 0.20 100.09 0.35 20
C12Oleth-10548 h0.81 1.06 400.47 0.75 300.34 0.60 20
C12240.49 0.83 300.30 0.59 200.30 0.51 20
C1240.09 0.35 200.06 0.25 100.04 0.20 10
C13WP 48 h0.17 0.43 200.04 0.20 100.11 0.37 20
C13240.04 0.20 100.00 0.00 000.04 0.20 10
C1340.04 0.20 100.02 0.15 100.09 0.35 20
C14DW 48 h0.45 0.65 200.32 0.63 300.21 0.46 20
C14240.21 0.51 200.15 0.42 200.36 0.67 30
C1440.06 0.32 200.13 0.45 200.04 0.29 20
C15Saline 48 h0.26 0.49 200.06 0.25 100.06 0.25 10
C15240.11 0.31 100.06 0.25 100.11 0.31 10
C1540.09 0.35 200.02 0.15 100.06 0.32 20
Table 4. The summary of results in Japanese standard
ChemSampleConc. (%)hourEvaluation 2 h afterEvaluation 24 h afterEvaluation 48 h after
MeanSDMaxMinMeanSDMaxMinMeanSDMaxMin
C01SLS0.548 h0.43 0.35 100.67 0.50 200.47 0.34 10
C01240.49 0.51 200.69 0.50 200.44 0.45 20
C0140.07 0.18 0.500.11 0.23 100.06 0.17 0.50
C02SLS0.348 h0.32 0.30 100.63 0.53 200.36 0.36 10
C02240.21 0.29 100.46 0.44 200.26 0.33 10
C0240.05 0.16 0.500.07 0.18 0.500.05 0.16 0.50
C03SLS0.148 h0.10 0.20 0.500.21 0.29 100.14 0.25 10
C03240.13 0.24 100.21 0.29 100.13 0.22 0.50
C0340.10 0.20 0.500.00 0.00 000.02 0.10 0.50
C04PG5048 h0.34 0.28 100.26 0.29 100.16 0.24 0.50
C04240.23 0.39 200.13 0.22 0.500.05 0.16 0.50
C0440.02 0.10 0.500.03 0.12 0.500.05 0.16 0.50
C05PG3048 h0.32 0.37 200.24 0.25 0.500.17 0.26 10
C05240.14 0.23 0.500.11 0.21 0.500.10 0.20 0.50
C0540.01 0.07 0.500.00 0.00 000.04 0.14 0.50
C06SL248 h0.48 0.39 200.39 0.33 100.24 0.29 10
C06240.45 0.33 100.47 0.34 100.37 0.32 10
C0640.38 0.49 200.17 0.24 0.500.09 0.22 10
C07SL148 h0.21 0.27 100.15 0.23 0.500.07 0.18 0.50
C07240.27 0.27 100.26 0.25 0.500.16 0.24 0.50
C0740.18 0.30 100.09 0.19 0.500.05 0.16 0.50
C08IPM10048 h0.10 0.20 0.500.05 0.16 0.500.10 0.20 0.50
C08240.02 0.10 0.500.02 0.10 0.500.05 0.19 10
C0840.02 0.10 0.500.00 0.00 000.04 0.14 0.50
C09BC0.148 h0.36 0.34 100.29 0.29 100.23 0.31 10
C09240.38 0.51 200.30 0.40 200.19 0.27 10
C0940.13 0.22 0.500.01 0.07 0.500.02 0.10 0.50
C10BC0.0548 h0.26 0.29 100.23 0.29 100.19 0.27 10
C10240.16 0.24 0.500.15 0.34 200.09 0.22 10
C1040.04 0.14 0.500.04 0.14 0.500.02 0.10 0.50
C11Oleth-101048 h0.16 0.24 0.500.18 0.24 0.500.06 0.17 0.50
C11240.14 0.27 100.05 0.16 0.500.12 0.21 0.50
C1140.05 0.16 0.500.02 0.10 0.500.03 0.12 0.50
C12Oleth-10548 h0.30 0.40 200.18 0.26 100.14 0.23 0.50
C12240.18 0.28 100.12 0.21 0.500.14 0.23 0.50
C1240.03 0.12 0.500.03 0.12 0.500.02 0.10 0.50
C13WP 48 h0.07 0.18 0.500.02 0.10 0.500.04 0.14 0.50
C13240.02 0.10 0.500.00 0.00 000.02 0.10 0.50
C1340.02 0.10 0.500.01 0.07 0.500.03 0.12 0.50
C14DW 48 h0.18 0.24 0.500.14 0.25 100.10 0.20 0.50
C14240.09 0.19 0.500.06 0.17 0.500.15 0.25 10
C1440.02 0.10 0.500.04 0.14 0.500.01 0.07 0.50
C15Saline 48 h0.12 0.21 0.5 0.03 0.12 0.500.03 0.12 0.50
C15240.05 0.16 0.500.03 0.12 0.500.05 0.16 0.50
C1540.03 0.12 0.500.01 0.07 0.500.02 0.10 0.50
Figure 2.

New standard. BC, benzalkonium chloride; PG, Propylene glycol; SL, sodium laurate; SLS, sodium lauryl sulfate.

Figure 3.

Japanese standard. BC, benzalkonium chloride; PG, Propylene glycol; SL, sodium laurate; SLS, sodium lauryl sulfate.

At 24 h after patch removal, there was no significant association between the incidence of skin reaction and application time (4, 24 or 48 h) for white petrolatum or saline solution (negative controls) or for distilled water (a solvent control according to the new criteria for evaluating skin irritation); however, there was a significant association between the incidence of skin reaction and application time for all samples except the isopropyl myristate (IPM) sample, for which the incidence of reaction was low at all three application time points. There was no significant difference in the incidence of skin reaction after applications of 24 and 48 h, except for the 30% propylene glycol (PG) aqueous solution. Regarding the incidence of skin reaction as determined by the standard Japanese criteria, most results were identical to those obtained using the new criteria for all three application times, although there was a significant difference in the incidence of skin reactions after 24 and 48 h of application for the 30% PG aqueous solution, the SL (1%) aqueous solution, and oleth-10 (10%; χ2-test, P < 0.05).

According to the new criteria, the mean irritation score for SLS was similarly high after 24 and 48 h of application, at each concentration of SLS. However, the scores after only 4 h of application were markedly lower. For all concentrations of PG, the mean irritation score increased with increasing application time. For all concentrations of SLS there was little difference in mean irritation score as a result of application time at 2 h after patch removal. For an application time of 4 h, the mean irritation score tended to decrease with time. However, after 24 and 48 h of application, the decrease was comparatively slight. Mean irritation score was low at all application times for IPM . For benzalkonium chloride, the mean irritation scores were similarly high after 24 and 48 h of application, as was the case with SLS. However, the mean irritation score after 4 h of application was markedly lower. Trends for the mean Sugai irritation score,[17] which is based on the Japanese criteria, were much the same as those calculated using the new criteria.

Discussion

Using both new and standard criteria, we were able to detect irritation after 24 and 48 h of patch application. However, it was difficult to detect irritation after 4 h of application because the incidence of irritation was low. There was a significant difference in the incidence of irritation after 24 and 48 h of application for PG (30%) solution, SL (1%) solution and oleth-10 (10%), substances for which irritation levels were low. However, no such difference was found for substances that cause greater irritation, that is, there was no marked difference in the incidence of irritation by application time. Plasters attached for 48 h caused a rash in some volunteers (Fig. 4), which indicates that leaving patches in place for this length of time places a greater burden on people undergoing the test.

Figure 4.

The left upper back was used for 48-h patch application, and the right rash is clearly more severe on the left upper back.

For patch application times of 24 and 48 h, the incidence of skin irritation reaction at 24 h after was relatively higher or almost the same, as compared to 48 h after patch removal. This indicates that skin irritation reactions can be examined at 24 h after patch removal.

The samples used in this study are all commonly available. In particular, SLS is used as a positive control in research on the evaluation of primary skin irritation.[17-19] These reports describe the concentration of SLS, as well as the application time, observation period and part of the body to which SLS is applied. Many reports suggest a concentration of between 0.1% and 2% for use in studies; SLS is usually applied to the skin using a patch for 4, 23, 24 or 48 h.

Testing with an application time of 4 h was developed to predict acute skin irritation to chemical substances and to rank substances according to the resulting irritation. Because the main objective of this method is to detect skin irritation and the toxicity of chemical substances, samples are usually used at high concentrations and short exposure periods. The concentration of SLS – used as a positive control in such tests – is 20%, and a positive rate of at least 30% is required for the test to be valid.[10] In clinical practice, the 48-h patch test is usually used, although 23- or 24-h patch tests can also be used for evaluation of skin irritation.

In many reports, an observation period of 1, 24 or 48 h after patch removal is selected, irrespective of the application time, with patches usually attached to the upper back, forearmor upper arm. Löffler et al. performed a 48-h patch test with various SLS concentrations and reported that with a 0.5% SLS aqueous solution there was a strong correlation between reactions on the upper back and on the forearm, and that the reactions on the forearm were more noticeable than those on the upper back. They also reported that a 0.5% SLS aqueous solution, applied to the forearm for 48 h, was useful for evaluation of skin irritation and that mild irritation values were observed at 24 h after patch removal.[5]

In the present study using SLS 0.5% aqueous solution, 42.6% (20/47) of volunteers had a clear positive response (≥3 according to the new criteria for skin irritation and + or higher according to the standard Japanese criteria) at 24 h after patch removal on the 24-h patch test; 40.4% (19/47) had such a response on the 48-h patch test. Although there are differences between the upper back and forearm, the results obtained in the present study agree with those of earlier studies, suggesting that the conditions in the present study allow mild skin irritations to be accurately detected. There were no important differences in the results on irritation reaction obtained according to the standard Japanese criteria for determining skin irritation and the new criteria.

In conclusion, a patch application time of 24 h is sufficient to detect primary skin irritation from irritants in cosmetics and other common household products. In addition, we found that skin irritation reactions were strongest at 24 h after patch removal and that the reaction tended to be weaker at 48 h after patch removal. These results suggest that, for optimum results, patch testing to evaluate irritants can be performed by means of a 24-h patch test with a follow-up reading at 24 h after patch removal. In addition, an application time of 24 h places less of a burden on patients than a 48-h patch test.

Acknowledgment

This study was performed by the Joint Research Fund of the Japanese Society for Dermatoallergology and Contact Dermatitis.

Ancillary