**6. References**

66 Contact Dermatitis

or low molecular weight as acyclovir may give doubtful results in sensitized patients, possibly due to poor penetration of this substances through the epidermis. Scratch–patch testing, by compromising epidermal barrier function, enables enhanced penetration of substances into the skin [**35-37**]. The method is performed by causing mechanical injury to the epidermis with a sterile skin prick lancet in order to compromise the stratum corneum, which represents the most important barrier limiting hapten penetration. The test reactions are usually read after D2 and D3, when possible, also after D4 and D7. The method of grading a positive scratch–patch test is identical to that used for conventional patch testing with no differences. It can be used for many drugs: low molecular wheight molecules (e.g. *β*-blockers, antiviral drugs etc.) and also high molecular wheight

Contact allergy to topical *β*-blockers is a well-recognized side-effect of glaucoma treatment [**38-41**]. Sensitization may be singly to agents such as timolol, befunolol, levobunolol, or,

A closed patch test, usually used in clinical practice for the diagnosis of allergic contact dermatitis, is often sufficient to show *β*-blockers contact allergy. However, there may be difficulties in obtaining positive patch tests to *β*-blockers, as showed in earlier reports [**42- 43**]. Poor penetration through intact skin on the back, where patch testing is normally

Topical antiviral drugs are frequently used, but although repeated applications can lead to contact reactions **[44-45]**, adverse cutaneous reactions are not commonly observed. Allergic contact dermatitis caused by acyclovir is rare, with only 20 studies reported **[46-48].** Because of the doubtful reactions with antiviral, especially acyclovir, and in view of the suggestive clinical history, we recommend the scratch–patch test followed by repeated open

Heparin is a sulfated glycosaminoglycan with anticoagulant properties. It is usually injected intravenously or subcutaneously but is also available for topical application. Cutaneous

We report a case of patch-test-negative allergic contact dermatitis, diagnosed by scratch patch testing, from a gel containing heparin. Allergic reaction to subcutaneously injected heparins is not a rare occurrence **[51-52]** but there are only a few reports of contact

In cases of suspected contact allergy, when conventional closed patch test shows negative or doubtful results, scratch–patch testing should be considered. We recommend, after performing scratch-patch test, to execute a ROAT to be sure the drug can be applied

allergic reactions due to subcutaneously injected heparin have been reported **[49-50].**

molecules (e.g. heparin etc.):

applied, may be a factor.

**5.3.2 Antiviral drugs** 

application test (ROAT).

dermatitis from topical heparin **[53].**

**5.3.3 Heparin** 

safely.

more rarely, to multiple *β*-blockers in a single patient.

**5.3.1** *β***-blockers** 


Topical Delivery of Haptens: Methods of Modulation of the

Contact Dermatitis 1997:10:1113–6.

Dermatitis 1993:28:41–2.

2001:12:217–9.

2001:44:369.

2002:46:313–4.

2001:44:265–9.

51.

Dermatitis 1990:22:61–2.

Contact Dermatitis 2003:49:155–7.

Cutaneous Permeability to Increase the Diagnosis of Allergic Contact Dermatitis 69

[34] Holdiness MR. Contact dermatitis to topical drugs for glaucoma. Am J Contact Dermat

[35] Nino M, Suppa F, Ayala F, Balato N. Allergic contact dermatitis due to the beta-blocker

[36] Katoh N, Kanzaki T. Contact dermatitis due to befunolol hydrochloride eyedrops.

[37] Van der Meeren H L, Meurs P. Sensitization to levobunolol eyedrops. Contact

[38] Quiralte J, Florido F, Saenz de San Pedro B. Allergic contact dermatitis from carteolol

[39] Corazza M, Virgili A, Mantovani L, Taddei Masieri L. Allergic contact dermatitis from

[40] O'Donnell B F, Foulds I S. Contact allergy to beta-blocking agents in ophthalmic

[41] Sánchez-Pérez J, Jesús Del Río M, Fernández-Villalta M J, García-Díez A. Positive use

[42] Nino M, Balato N, Di Costanzo L, Gaudiello F. Scratch-patch test for the diagnosis of allergic contact dermatitis to aciclovir. Contact Dermatitis 2009:60:56–7. [43] Serpentier-Daude A, Colet E, Didier A F et al. Contact dermatitis to topical antiviral

[44] Holdiness M R. Contact dermatitis from topical antiviral drugs. Contact Dermatitis

[45] Goh C. Compound allergy to Spectraban 15 lotion and Zovirax cream. Contact

[46] Vernassiere C, Barbaud A, Trechot P H et al. Systemic acyclovir reaction subsequent to

[47] Lammintausta K, Makela L, Kalimo K. Rapid systemic valaciclovir reaction subsequent

[48] Maroto-Iitani M, Higaki Y, Kawashima M. Cutaneous allergic reaction to heparins:

[49] Hohenstein E, Tsakiris D, Bircher AJ. Delayed-type hypersensitivity to the ultra-

[50] Jappe U, Juschka U, Kuner N, Hausen BM, Krohn K.. Fondaparinux: a suitable

[51] Schindewolf M, Ludwig RJ, Wolter M, Himsel A, Zgouras D, Kaufmann R, et al.

heparinoids? A study of 7 cases. Contact Dermatitis 2004;51:67–72.

heparin. J Eur Acad Dermatol Venereol 2008;22:378–80.

to aciclovir contact allergy. Contact Dermatitis 2001:45:181.

acyclovir contact allergy: which systemic antiviral drug should then be used?

subcutaneous but not intravenous provocation Contact Dermatitis 2005;52:228–30.

low-molecular-weight heparin fondaparinux. Contact Dermatitis 2004;51:149–

alternative in cases of delayed-type allergy to heparins and semisynthetic

Tolerance of fondaparinux in patients with generalized contact dermatitis to

test in contact dermatitis from betaxolol hydrochloride. Contact Dermatitis

cross-reacting β-blocking agents. Contact Dermatitis 1993:28:188–9.

and timolol in eyedrops. Contact Dermatitis 2000:42:245.

preparations. Contact Dermatitis 1993:28:121–2.

drugs. Ann Dermatol Venereol 2000:127:191–4.

befunolol in eyedrops, with cross-sensitivity to carteolol. Contact Dermatitis


[18] Mauro T, Hollerann WM, Grayson S, Gao WN, Man MQ, Kriehuber E, et al. Barrier

[19] Rougier A, Lotte C, Corcuff TP. Relationship between skin permeability and cornecyte

[20] Berardesca E, Maibach HI. Racial differences in skin pathophysiology. J Am Acad

[21] Menon GK, Elias PM, Feingold KR. Integrity of the permeability barrier is crucial for manteinance of the epidermal calcium gradient. Br J Dermatol 1994;130:139-47. [22] Surber C, Davis AF. Bioavailability and Bioequivalence of Dermatological

[23] Hauck WW. Bioequivalence studies of topical preparations: statistical considerations.

[24] Skelly JP, Shah VP, Maibach HI. FDA and AAPS report of workshop on principles and

[25] Shah VP, Elkins J, Hanus J, Noorizadeh C, Skelly JP. In vitro release fo hydrocortisone from topical preparations and automated procedure. Pharm Res 1991;8:55-9. [26] Davis AF, Gyurik RJ, Hadgraft J. Formulation strategies for modulating skin

[27] Patil S, Singh P, Szolar-Platzer C, Maibach HI. Epidermal enzymes as penetration enhancers in transdermal drug delivery? J Pharm Sci 1996;85:249-52. [28] Mitragotri S. Synergistic effects of enhancers for transdermal drug delivery. Pharm Res

[29] Tsai JC, Guy RH, Thornfeldt CR, Gao WN, Feingold KR, Elias PM. Metabolic

[30] Johnson ME, Mitragotri S, Patel A, Blankschtein D, Langer R. Synergistic effects of

[31] Choi EH, Lee SH, Ahn SK, Hwang SM. The pretreatment effect of chemical skin

[32] Singh J, Maibach HI. Transdermal delivery and cutaneous reactions. In: Kenneth AW,

[33] Jappe U, Uter W, Menezes de Pádua C A, Herbst R A, Schnuch A. Allergic contact

surveillance data 1993–2004. Acta Derm Venereol 2006:86:509–514.

extracellular lipid processing. Arch Dermatol Res 1998;290:215-22.

Formulations. New York: Marcel Dekker, Inc;2002..p401-498.

21.

Dermatol 1996;34:667-72.

2000; 17:1354-9.

Int J Dermatol 1992;31 (suppl. 1):29-33.

inhibitors. J Pharm Sci 1996;85:643-8.

Pharm Sci 1996;85:670-9.

Physiol 1999;12:326-35.

1995;132:571-9.

and bioequivalence. Pharm Res 1987;4:265-71.

Formulations. New York: Marcel Dekker, Inc;2002.

recovery is impeded at neutal pH, independent of ionic effects: implications for

size according to anatomic site, age and sex in man. J Soc Cosmet Chem 1988;39:15-

Formulations. In: Kenneth AW, editor. Dermatological and Transdermal

practices of in vitro percutaneous penetration studies: relevance to bioavailability

permeation In: Kenneth AW, editor. Dermatological and Transdermal

approaches to enhance transdermal drug delivery. Effect of lipid synthesis

chemical enhancers and therapeutic ultrasounds on transdermal drug delivery. J

penetration enhancers in transdermal drug delivery. Skin Pharmacol Appl Skin

editor. Dermatological and Transdermal Formulations. New York: Marcel Dekker, Inc;2002. 34. Korting HC, Stolz W, Schmid MH, Maierhofer G. Interaction of liposomes with human epidermis reconstructed in vitro. Br J Dermatol

dermatitis due to beta-blockers in eye drops: a retrospective analysis of multicentre


**5** 

**Progress on the Development of Human** 

*In Vitro* **Assays for Assessment of the** 

**Sensitizing Potential of a Compound:** 

*Department of Dermatology, VU University Medical Centre, Amsterdam,* 

Contact dermatitis is a common health problem, which affects both men and women and accounts for 85-90% of all skin diseases. Two main types of contact dermatitis can be distinguished, according to the patho-physiological mechanisms involved, i.e. allergic and irritant contact dermatitis. Allergic contact dermatitis requires the activation of antigen specific (i.e. acquired) immunity leading to the development of effector T cells, which mediate skin inflammation (Nosbaum et al., 2009; Saint-Mezard et al., 2004). Irritant contact dermatitis is due to inflammatory and toxic effects caused by exposure to xenobiotics activating an innate local inflammatory reaction (Mathias and Maibach, 1978; Nosbaum et al., 2009). Identification of a potential sensitizer and its distinction from an irritant substance (non-sensitizer) currently completely relies on animal testing. The mouse Local Lymph Node Assay (LLNA) is the most frequently used and accurate test with regards to relevance (predictive capacity) and reliability (reproducibility within and between laboratories) in distinguishing a sensitizer from a non-sensitizer (Basketter et al., 2007; Gerberick et al., 2005; Kimber, 2002). This is closely followed by the Guinea pig maximization test (Basketter and Scholes, 1992). In Europe as from 2013 animal testing of cosmetic products will be prohibited (Directive 76/768/EEC), while the implementation of the REACH (Registration, Evaluation and Authorization of Chemicals) legislation (European Regulation 2006) will result in an increased demand for risk assessment of nearly 30,000 chemicals already marketed in the EU. Moreover, the replacement, reduction, and refinement of the use of test animals in general is now strongly advocated. Therefore, there is an urgent need for reliable *in vitro* assays, which are able to distinguish sensitizers from non-sensitizers. This chapter describes the progress being made to develop a battery of assays, which mimics human sensitization *in vitro* and therefore

which may in the future be able to replace the use of test animals.

In order to develop such a battery of assays it is important to first understand the different *in vivo* events which occur during sensitization. The skin functions as a barrier protecting an individual from dehydration, mechanical trauma, irradiation, microbial insults, and

**1. Introduction** 

**Breaking Down the** *In Vivo* **Events** 

Susan Gibbs and Krista Ouwehand

*The Netherlands* 

[52] Nino M, Patruno C, Zagaria O, Balato N. Allergic contact dermatitis from heparincontaining gel: use of scratch patch test for diagnosis. Dermatitis 2009: 20: 171– 172.
