Irritancy Testing Protocols

Direct irritation may thus be dehned as an adverse effect of chemicals directly applied to the skin that does not involve prior sensitization and thus initiation by an immune mechanism. Irritation is usually assessed by a local inflammatory response characterized by erythema (redness) and/or edema (swelling). Other responses may be present that do not elicit inflammation such as an increase in skin thickness. Irritant reactions may be classified as acute, cumulative, traumatic, or pustular. However, two classifications are generally used by toxicologists. Acute irritation is a local response of the skin usually caused by a single agent that induces a reversible inflammatory response. Cumulative irritation occurs after repeated exposures to the same compound and is the most common type of irritant dermatitis. 

There are several types of irritancy testing protocols that are used to comply with federal and international safety regulations. The classic Draize test was developed in 1944 to measure acute primary irritation. The test compound is applied in an occluded fashion to a clipped area of abraded and intact skin of at least six albino rabbits and evaluated 24 hr and 72 hr after patch removal. The degree of erythema and edema, ranging from one to four, is recorded to reflect severity of the irritation. Because these tests are occluded, irritancy is potentiated due to hydration, which reduces the skin barrier. The Draize test may be modified to assess sensitization by preexposing animals to a sensitizing dose of the study chemical and then rechallenging the animals at a later date to illicit the immune-mediated response. 

In addition to the above testing protocols, the local lymph node assay (LLNA) has been validated and accepted to assess the skin sensitization potential of chemicals in animals. This does not replace the guinea pig maximization test but is considered to be an equivalent. This in vivo method helps to reduce the number of animals used for contact sensitization activity. This test is based on the principle that sensitizer can induce a primary proliferation of lymphocytes in the lymph node draining the site of chemical application. This provides a quantitative measurement in which the proliferation is proportional to the dose applied. 

---

A study on human abdominal skin showed that fluorescent dye embedded in a bilayer made from gel-state PC was taken up by the skin,24 but the dye penetrated only into stratum corneum. The results of this experiment are illustrated in Figure 23.2. Similar results were reported also by others.22,23 35 The tolerance of skin of 20 volunteers to the gel-state PCs was studied in comparison to some common emulsifiers. Nine emulsifiers and gel-state PC were tested in the Duhring Chamber using test protocol of Frosch and Kligman.36 Based on scaling and erythema, gel-state PCs were the only substances that showed no irritation potential.37 The results are shown in Figure 23.3. Reviewed recently were also the toxicological aspects of the use of gel-state PC in a topical application.7... 

More that 5300 test samples have been studied in the Skintex system, including petrochemicals, agrochemicals, household products, and cosmetics. The reproducibility with standard deviations of 5-8% is excellent. New protocols applicable to very low irritation test samples and alkaline products have increased the applicability of this method. Skintex validation studies resulting in an 80-90% correlation to the Draize scoring have been reported by S.C. Johnson Son and the Eood and Drug and Safety Center. 

All of these skin equivalent methods permit higher concentrations of test samples to be studied. However, dilutions are still necessary when, based on the physical chemistry of the test sample, the chemical structure may be responsible for irritation. Many protocols and endpoints have been evaluated as predictive of eye or skin irritation. 

The EpiOcular model uses normal human-derived epidermal keratinocytes, which form a stratified squamous epithelium morphologically similar to that found in the cornea [36]. The main endpoint evaluated is cell viability measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Thiazolyl blue EINECS number 206-069-5, CAS number 298-93-1), into a blue formazan salt that is quantitatively measured after extraction from tissues. Two different protocols exist, the EpiOcular ET50 mainly developed for the testing of surfactants and surfactant-based mixtures, and the EpiOcular Eye Irritation Test (EIT) developed for a variety of chemistries. 

Spoo J, Wigger-Alberti W, Bemdt U, et al. Skin cleansers three test protocols for the assessment of irritancy ranking. Acta Derm Venereol. 2002 82 13-7. 

The development in recent years of noninvasive instrumental techniques has considerably increased the level of discrimination between products or surfactants. Significant differences in terms of interaction of products with skin surface are now detected much earlier than clinical signs of irritation. Different test protocols have been described in the literature [86] and have been developed in order to induce no or minimal clinical irritation and compare the effect of siufactants by means of instrumental measurements. Transepidermal water loss (evaporimetry), skin capacitance/conductance (skin surface electrical measurements), and vascular status (laser Doppler flowmetry) measurements seem among the most sensitive bioengineering methods for such a purpose. They assess the effect of the surfactants on alteration of the skin barrier function, skin surface hydration, and microvessels blood flow, respectively. 

By using two distinct test protocols, Paye et al. [133] observed a different ranking in terms of skin irritation potential among several personal cleansing products. A modification of the ranking of products by changing test conditions is, however, rarely observed and should be specific to both the application conditions and products used in the test. 

P4HB and P3HB-4HB have been evaluated in preclinical tests reconunended by the FDA for medical devices. These tests include cytotoxicity, sensitization, irritation and intracutaneous reactivity, hemocompatibility, and implantation. Thus for example, P4HB films and sutures were subjected to a complete series of biocompatibility test protocols that were performed in accordance with the FDA s GLP regulations as set forth in 21 CFR, part 58, as well as ISO 10993-1. The test results confirmed that P4HB is nontoxic and biocompatible (Martin DP, personal communication). 

Katoh M, Hamajima F, Ogasawara T, Hata K. 2010. Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol. J. Toxicol. Sci. 34(3) 327. 

Animal tests were commonly used in the past, also according to the Draize protocol [357,372]. However, such tests have been questioned for several reasons. In addition to the public concern about animal welfare, animal tests are conducted under conditions and at concentrations quite different from those that occur in practice, as in the case of eye irritancy. The effects of surfactants on the skin cannot be compared when the results have been obtained through different protocols and even results obtained by the same method are not comparable if conditions differ. 

Toxic responses in the eye can result from direct topical ocular exposure of drugs from direct installation into the eye and also from dermal products which patients may accidentally get into their eyes. Until recently, the Draize rabbit eye test using three rabbits has served as the major protocol to assess the irritancy potential of topically applied substances. 

A number of industrial chemicals were screened using AFNOR protocols with rabbits as the test systems. Information on the appearance and irritancy potential of different chemicals (and the classification thereof) offers valuable clues to the worker to avoid skin injury (Table 16-4). Certain drugs normally used by humans can cause adverse effects on eyes. The types of ocular disturbance are blurring of vision and diplopia, impairment of visual acuity, yellow vision (xanthopsia), corneal opacities, and lenticular opacities and drugs producing or precipitating the formation of cataract, which have been implicated with the use of candidate drugs. 

The EpiOcular EIT underwent prevalidation and validation studies. The method is based on the use of a single exposure time and has separate protocols for liquid and solid test substances. A test substance is classified as an irritant (GHS Cat. 1 or 2), if the tissue viability is <60 %, and non-irritant (non-classified) if the viability is >60 %. The prediction model was developed based on a training set of 60 substances (of a range of chemical classes) further challenged with the testing of 52 additional substances, which showed high sensitivity, and good specificity and concordance [37]. Based on these positive results, a prevalidation study was... 

---