Repeat Application Irritation Patch Tests

PURPOSE AND RATIONALE Inflammation produced late in the induction phase of sensitization tests without positive responses at challenge is cumulative irritation. The HRIPT for skin allergy was modified to evaluate skin irritation. As with single-application patch tests, many investigators developed their own version of the repeat application patch test. 

Kligman and Wooding (1967) applied the Litchfield and Wilcoxon probit analysis to cumulative irritation testing with calculation of IT 50 and ID 50 values. Their early work forms the basis for the 21-day cumulative irritation assay, which is currently widely used. 

The cumulative irritation assay was developed to compare antiperspirants, deodorants, and bath oils to provide guidance for product development. 

A 1-in square of Webril is saturated with liquid, or up to 0.5 g of viscous substances, and applied to the surface of the pad to be applied to the skin. The patch is applied to the upper back and sealed in place with occlusive tape. The patch is removed after 24 h, the area is evaluated and a fresh patch applied. The procedure is repeated daily for up to 21 days. Lanman et al. (1968) increased the sensitivity of the assay by increasing the number of test subjects from 10 to 24. 

The ID 50, as described by Kligman and Wooding (1967), was used to evaluate and statistically compare test materials. 

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Organophosphate Ester Hydraulic Fluids. Repeated application of a patch treated with 0.2 pL of Skydrol 500B-4 for 5 weeks (3 times/week) resulted in mild cumulative erythema confined to the contact site in 14 of 53 human test subjects, beginning with the third dose during the first week. No evidence of immediate primary dermal irritation was observed (Monsanto 1980). 

NMP produced no skin irritation with patch testing for 24 hours in 50 volunteers. A few mild transient reactions were noted after repeated application. There was no evidence of contact sensitization. 

Objective irritation is defined as nonimmunologically mediated, localized inflammation of the skin, usually resulting from contact with a substance that chemically damages the skin.2,9 The exact mechanism is unknown, and it is likely that both endogenous and exogenous factors are involved. In vivo predictive testing in animals (e.g., modified Draize test, repeated application patch tests,... 

Many industries regularly conduct repeat insult patch tests on human volunteers to evaluate topical irritancy. Groups of human volunteers are patched with test substance. One to five concentrations can be tested simultaneously, a wide enough range to yield results relevant to the usage. Cumulative skin irritancy is measured by applying patch applications each day for 3 weeks. Skin irritation is usually assessed visually, but blood flow and skin temperature can be measured objectively by laser Doppler flowmetry, ultrasound Doppler, heat flow disk measurement, sensitive thermocouple devices, or noncontact infrared radiative techniques. In these tests, dose-response curves can be obtained. Skin thickness can be measured with calipers as a measure of edema formation. 

The types of tests used to assess skin irritation potential of ingredients or products are many and varied. Tests on animals of different species are used to assess safety and are usually blunt tools. Rabbits, guinea pigs, and humans are frequently used species. Fluman in vivo tests of products or solutions of ingredients applied under patches or in plastic or glass chambers have been used. Arm or hand immersion or repeat applications to sensitive areas (e.g., the inside of the forearms or the cheeks) are also common test sites. 

The Buehler test (Buehler 1964) also employs topical application of the test material. An absorbent patch or vehicle alone is placed on the shaved flanks of 10-20 guinea pigs. Test concentration varies from undiluted to usage levels. A concentration that produces slight erythema is optimum and is selected based on an irritancy screen conducted in other animals. This procedure is repeated 7 days and 14 days after the initial exposure. Two weeks after the last induction patch, animals are challenged with patches saturated with a non-irritating concentration of test material and with the vehicle. After 6 h, the patch is removed and the area depilated. Test sites are visually evaluated 24 h and 48 h after patch removal. Animals developing erythematous responses are considered sensitized (if irritant control animals do not respond). 

Patch testing with serial dilution of nickel sulfate is sometimes used to gain more information on the degree of sensitivity and to discriminate between allergic reactions and irritant ones (Andersen et al. 1993 Wahlberg 1995). Open tests to study the concentration threshold have been carried out with nickel sulfate or chloride as single or repeated applications (Menne and Calvin 1993 Allenby and Basketter 1994). Intracutaneous testing with nickel sulfate is used at some centres (Moller 1989). 

Such tests are suitable for identifying highly irritant material, not for comparing irritancy potential of different products. Repeated open application tests are also frequently used to verify the clinical significance of allergic patch test reactions [55]. 

Repeat Animal Patch (RAP) test - compares irritation potential of surfactants. Solutions were applied to the clipped back of immobilized albino mice with a saturated cotton-tipped applicator. The test site was covered with a rubber dam to prevent evaporation. This process was repeated seven times at intervals of 10 min. The skins were evaluated microscopically for epidermal erosion. 

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