University of Groningen. P-phenylenediamine Bijkersma-Pot, Laura

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1 University of Groningen P-phenylenediamine Bijkersma-Pot, Laura IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2014 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Bijkersma-Pot, L. (2014). P-phenylenediamine: Insights in penetration, sensitization and elicitation [S.l.]: [S.n.] Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 General discussion and future perspectives CHAPTER L.M. Pot Department of Dermatology, University Medical Center Groningen, University of Groningen, The Netherlands 109

3 General discussion and future perspectives 110 The immunogenic potential of p-phenylenediamine (PPD) has been recognized since the 19 th century 1, 2 and PPD is still an important allergen in scientific literature. Based on the local lymph node assay (LLNA) PPD is regarded as an extreme sensitizer 3 with the potency to sensitize 100% (n = 24) of the humans exposed to 10% PPD, as shown by the historical human maximization test published in 1966 by Kligman. 4 Currently, this extreme sensitizing potency of PPD is clearly reflected when individuals - often children and adolescents - are exposed to PPD containing temporary black henna tattoos. In contrast to natural red henna (Lawsonia inermis) which is considered relatively safe and rarely sensitizing, 5 black henna is an artificial mixture of red henna and PPD, although some mixtures do not even contain the red henna component. 6,7 In my opinion these tattoos can be considered as a modern, although unintentional, human maximization test, because some of the black henna mixtures have been shown to contain even higher percentages of PPD than used in the - nowadays considered unethical - human maximization test. It is therefore highly understandable that the application of PPD in temporary henna tattoos is prohibited in Europe and the United States. 9,10 In fact, in Europe PPD is currently only allowed to be used in hair dyes, with an upper concentration limit of 4% (or 2% as final on-head concentration after being mixed to the oxidizing agent). 11 Although hair dyeing is still considered the most common cause of PPD sensitization, 12 other sources including the temporary black henna tattoo as well the diagnostic patch test are increasingly reported. 5,13 Often, the source of exposure is unknown; a large study (N = 3307) showed that in about 44% of the subjects diagnosed to be sensitized to PPD, the probable source of exposure could not be identified. 14 In addition, a positive patch test to PPD might also be due to cross-reactivity. Commonly reported cross-reacting allergens are structurally related chemicals such as the hair dye precursor p-toluenediamine (PTD), the sunscreen component para-aminobenzoic acid (PABA), other azo dyes frequently used in textiles (Disperse Orange 3, Disperse Yellow 3, Disperse Blue), local anaesthetics such as benzocaine and black rubber N-isopropyl-N -phenyl-p-phenylenediamine; IPPD) In fact, a recent study by Thomas et al. (2014) suggested that the rate of cross-reactivity increases with increasing strength of the PPD patch test. 20 This should be taken into account when studying subjects which are strongly sensitized to PPD, especially in the absence of a clear history of exposure. The discussion on the extent of active sensitization to PPD by the diagnostic patch test is unresolved, with widely varying figures and opinions in the literature. 13,21-23 Late reactions to a patch test, i.e. de novo positive reactions after day 7-10 or later 24 might indicate active sensitization, although late reactions may also be inherent to the chemical (e.g. for

4 corticosteroids or metals) 25,26 or reflect a subject s low responsiveness. 27 A large prospective multicentre study performed by the German Contact Dermatitis Research Group (Deutsche Kontaktallergie-Gruppe; DKG) which found that PPD elicited late reactions in 1.5% of routine patch tests, considered this indicative of an actual risk of active sensitization and therefore the DKG decided to withdraw PPD from the baseline series in Germany in The European Society of Contact Dermatitis (ESCD) did not endorse this opinion. 2 In the context of active sensitization, Geier et al. (2013) assessed whether lowering the patch test concentration to 0.3% PPD pet. could be a safer alternative with still sufficient sensitivity, 29 since a study by the DKG already showed that lowering the patch test concentrations to 0.5%, 0.4% and 0.35% PPD pet. decreased the risk of active sensitization. Although 26% (32/123) of the subjects with a positive reaction to 1% PPD pet. did not elicit a positive reaction to the 0.3% PPD pet. patch test, the majority (69%) of these missed cases were not clinically relevant. Furthermore, with good quantitative parameters of diagnostic selectivity, i.e. the Reaction Index and Positivity Ratio, 30,31 the authors recommended replacement of the 1% PPD pet. by the 0.3% PPD pet. In addition, Søsted et al. (2006) showed that in a small population (N = 15) of subjects sensitized to PPD, 1/15 (6.7%) reacted to a 4 hours patch test containing 0.003% PPD in petrolatum, whereas 0.05% PPD in petrolatum increased this rate to 73% and a further increase in PPD concentration to 0.5%, resulted in 7% of positive elicitation response. 32 The high response rate to a PPD concentration of as low as 0.05% seems surprising, but this might be explained by the fact that the strength of the previous diagnostic patch test was not reported (except for one subject graded 2+ previously), and that this group may have consisted of strongly sensitized subjects for whom the elicitation threshold is assumed to be lower than for the weakly sensitized subjects. 33 Data from studies presented in this thesis contribute to this discussion of active sensitization by patch testing with 1% PPD in petrolatum. In chapter 3 the human in vivo penetration study learned us that after a short application of only 30 minutes of a 1% PPD in petrolatum patch, already significant amounts of PPD (90 mg PPD/g keratin) can be found on the skin s surface (0 micron depth). This actually corresponds to a level of 9 mass-% PPD of dry skin. Applying a 1% PPD petrolatum patch for 2 hours or even 3 hours (with refreshment of the patch every hour) resulted in PPD levels of 150 mg PPD/g keratin and 330 mg PPD/g keratin respectively, measured directly after removal of the patch. Furthermore it was shown that within the time frame of 27 h, approximately 330 mg PPD/g keratin had penetrated from the skin surface into the living epidermis and almost no PPD was left (17.2 mg PPD/g keratin compared to the detection limit of 13 mg PPD/ g keratin). In addition, a 23 h patch demonstrated that directly after removal of the patch, the amount of PPD in the skin approached the amount found after the 30 min application (i.e. 90 mg PPD/g keratin), implying that within the time span of 23 h, the PPD patch, or at least its layer closest to the skin surface, is depleted of PPD. 111

5 This calls into question the duration of the application of the diagnostic patch test (i.e. 4 hours), in addition to the previously discussed concentration of PPD. However, we acknowledge the fact that other factors such as practicability and standardization of application time may be much more decisive in the patch test procedure than single chemical depletion time. The high level of PPD penetration after application of 1% PPD in a petrolatum formulation (such as used for patch testing) was also shown in ex vivo experiments, as previously published by our group. 34 This study used pig skin in flow-through diffusion cells, to study the actual exposure levels of PPD, expressed as the Measured Exposure Level (MEL) which was calculated as the sum of PPD associated with the skin (dermis and epidermis including stratum corneum) and the receptor fluid. Interestingly, it was shown that under diagnostic patch test conditions (4 h application of a 1% PPD in petrolatum patch) the MEL is more than 10-fold higher than the MEL for hair dyeing conditions (2% PPD in hair dye formulation applied for 30 min with rinsing). When considering the applied doses for both conditions, i.e µg PPD cm -2 for the hair dye condition versus 400 µg PPD cm -2 for the patch test condition, 34 the fundamental difference between the dose applied and the dose actual becoming available for elicitation (the MEL) becomes even more evident. As demonstrated in chapter 6 of this thesis, in which available MEL data were examined against human elicitation responses in relation to applied dose and exposure time, we confirm that the MEL is a useful tool to better characterize thresholds of elicitation than the applied dose alone. In this context, it should be noted that the PPD TRUE test has an estimated MEL of almost a fifth of the MEL estimated for the 1% PPD in petrolatum patch test (45 µg PPD cm -2 compared to 205 µg PPD cm -2 ). 112 As described for PPD in chapter 2, studying penetration and haptenation is intriguing since PPD is unstable, leaving several intermediates with its own (often only partly- or unknown) penetration and haptenation characteristics. Complex auto-oxidation processes have also been described for other compounds such as the fragrances limonene, citronellol, linalool and geraniol Rüdback et al. recently described that not the parent compound citronellol, but its primary auto-oxidation products, the hydroperoxides, are the main sensitizers. 36 Similar to PPD, citronellol is not directly protein-reactive and therefore it has a very low sensitizing potency itself. As for PPD, auto-oxidation of citronellol renders the more sensitizing derivatives and hence, both chemicals are classified as pre-haptens. 36,39 However, when comparing the degradation processes of the parent compounds into the auto-oxidation derivatives, a striking difference is found in the rate of auto-oxidation. PPD has been shown to be degraded to 50% of its initial concentration after hours of air oxidation, 40 whereas as much as 10 weeks of air oxidation were needed to degrade 16% of the citronellol into its oxidation products. 36 This example emphasizes the instability of PPD and therefore the complexity of studying its immunogenic characteristics. Moreover, the fact that in daily life PPD is used in a hair dye

6 formulation containing other chemicals such as couplers and an oxidizing agent makes it even more challenging. Nevertheless, by reviewing papers published on penetration of PPD under different conditions (i.e. varying formulations, species and applied doses) we have shown that despite these differences, PPD penetration values after a short exposure time of around 30 minutes are quite similar. 41 The review further shows that the major proportion (>0%) of this low amount of PPD that penetrates the skin, will be acetylated to monoacetyl-ppd (MAPPD) and diacetyl- PPD (DAPPD) by the enzyme N-acetyltransferase 1 (NAT1). This results in an even smaller proportion (i.e. <20% of the already low amount of PPD that penetrates the skin) available for auto-oxidation and subsequent haptenation, yielding hapten-protein adducts that may induce sensitization. Whereas in vitro assays are able to predict the haptenation potential of oxidized PPD derivatives, in vivo correlation is still lacking since the exact fragments that are presented to the naïve T cells are unknown. 42 Furthermore, there may be many unknown (oxidative) PPD derivatives, the impact of which is unpredictable. This warrants further investigation as currently attempted by combining data from in vitro, in silico as well as in chemo assays. 43 In the in vivo penetration study presented in chapter 3 we demonstrated that in contrast to PPD, the acetylated PPD derivatives monacetyl-ppd (MAPPD) and diacetyl-ppd (DAPPD) and the oxidation product Bandrowski s base (BB) could not be identified within our experimental design. This is most probably caused by dilution, which occurs when these hydrophilic compounds enter the epidermis which has a relatively larger size and lower resistance to diffusion compared to the stratum corneum. 44,45 The experiments were performed within the existing safety margins, such as used in the diagnostic patch test procedure, but considering the high amount of PPD that penetrates the human skin after only a short application of the 1% PPD in petrolatum patch, we would recommend using skin equivalents for future investigations in order to prevent possible sensitization. One of our assumptions in this study was that - based on experimental data published in literature 46 - N-acetylation by NAT1 enzymes occurred in the viable epidermis, probably in particular in the basal cell layer. 47 It was therefore, that additional experiments with prolonged measurements were performed at a fixed depth of 30 micron, which has been shown to represent the viable epidermis at the volar forearm. 4 Interestingly, preliminary data from our group studying the localization of NAT1 in normal human skin suggests that this assumption should be revised, as NAT1 was shown to be abundantly present in the stratum corneum, in addition its perinuclear presence in the basal keratinocytes. (unpublished data) 113 By conducting an analysis of association as well as a meta-analysis we have shown in chapter 4 that common polymorphisms in genes encoding the detoxifying phase II enzymes glutathione-

7 114 S-transferase (GST) do not play a major role in sensitization to PPD and other xenobiotics. In fact, even when considering the combined high risk genotype GSTT1*0/GSTM1*0, to study a possible synergistic effect, no significant association with sensitization to PPD was found. This pointed to the conclusion that polymorphisms in GST genes have diseasemodifying, rather than disease-causing effect and that they only have a weak biologic impact. As described above, the identification of susceptibility loci in complex, multifactorial diseases such as allergic contact dermatitis is demanding, in view of the relatively low frequency in the population, the presence of genes with minor effects and ethnic differences. This has also been addressed in chapter 5, in our response to a comment of Westphal et al. in which we explain the differences found in the meta-analysis by the fact that GST have different modes of action and that detoxification of the studied xenobiotics cannot be solely attributed to the examined GST genes. 49 Consequently, as each individual has many single nucleotide polymorphisms (SNPs) that together create a unique DNA sequence, SNP-based genetic linkage analysis can be used to map disease loci, and hence determine disease susceptibility genes. The advances in SNP genotyping array technologies have made whole-genome association studies (WGAS) within reach. A single nucleotide polymorphism array is now a useful tool to study the whole genome for candidate genes. It is therefore, that we have established a consortium, comprising members of the European Society of Contact Dermatitis (ESCD), who will collect blood from subjects with a relevant sensitization to PPD or the closely related PTD 50 together with a short questionnaire to obtain information regarding exposure. The participating countries are Germany (Heidelberg, Jena, Trier), the Netherlands (Amsterdam, Groningen, Nijmegen), Portugal (Coimbra), Singapore, Spain (Barcelona) and Sweden (Malmö). Blood samples of a specific control group, consisting of a large sample of the general population in which exposure and PPD patch test data have been established carefully, will be used as controls. By means of this network, we will be able to collect a large enough case series (at least N = 2000) to draw reliable conclusions, based on the genetic variations found. The participating group is experienced and has done preliminary analytical work In addition, a European network will contribute to reduce the fragmentation in research investments. The elicitation response to PPD has previously been shown to be dose and exposure-time related. 32,55,56 This is not unique to PPD, as dose-response relationships in the elicitation phase also have been demonstrated for other compounds including nickel, 57 methylisothiazolinone, 5 isogeugenol 59 and many others. 60 However, the distinction made between the dose applied and the dose actually becoming available for elicitation, and the connection made to the elicitation response is unique. In chapter 6 it was shown that the elicitation dose needed to elicit allergic reactions in truly, but weakly PPD sensitized individuals, is between the dose becoming available by diagnostic patch testing and the dose becoming available by our model hair dye test exposure and that this dose

8 was different for each individual. Importantly, these differences in MEL only seemed to be of importance in patients who belong to the lower end of the patch test reactivity spectrum, given the fact that all previously tested ++ and +++ responders (in diagnostic patch testing), reacted to the 2%PPD hair dye test model. Thus, for this particular group of subjects, the + diagnostic PPD patch test seemed not to have a direct relevance for reactivity to a PPD concentration of 2% under simulated hair dye conditions. In these cases, the PPD diagnostic patch test only indicates sensitization and does not predict whether a contact allergic reaction will occur during daily life PPD exposure, i.e. hair dyeing. However, since only a limited number of subjects were available for this study, a larger number of subjects is needed to substantiate the results found thus far. The importance of the available dose (MEL) in evaluating contact allergic responses, regarding both the elicitation and the sensitization phase, has been reaffirmed in a report on quantitative risk assessment (QRA). 61 It was shown that for PPD the MEL under in use conditions is in close proximity to the no-expected-sensitization-induction-level (NESIL), indicating a high risk of induction of sensitization when used up to its maximal use concentration of 2%. This is in concordance with animal data (LLNA), which categorizes PPD in the range of extreme sensitizers. For resorcinol, another common ingredient in oxidative hair dyes, the MEL was shown to be much lower that the NESIL. This points to a negligible risk of induction of contact sensitization and confirms LLNA data categorizing resorcinol as only a moderate skin sensitizer. In line with the study described in chapter 6, chapter 7 describes the investigation of elicitation responses to a different hair dye molecule under use conditions in PPD allergic individuals. The hair dye used is based on a new, recently developed molecule, in which a methoxymethyl side chain was added to PPD, thereby forming methoxymethyl-ppd (ME-PPD). ME-PPD was developed since the most commonly used hair dye precursors PPD and PTD are the main allergens associated with hair dye related allergic contact dermatitis 62 and the demand for safe, permanent hair dye alternatives is high (author s experience from daily practice). It is a challenge to develop a new hair dye precursor, because good hair colouring performances often come together with a strong sensitizing potency, owing to the characteristic low molecular weight, the ability to penetrate the hair shaft and follicle and the strong protein reactivity. In the past, hydroxyethyl-p-phenylenediamine sulphate (HE-PPD), which has good colouring characteristics, had been shown to have less potential for crossreactivity compared to for example PTD in PPD sensitized subjects, but turned out to be a strong sensitizer according to the LLNA (EC3 value 0.57%; SCCS opinion on HE-PPD). 63 The recently developed ME-PPD holds the combination of a good hair colouring performance together with a moderate skin sensitizing potency (EC3 value LLNA 4.3%, compared to 0.1% and 0.17% for PPD and PTD) and could be promising for consumers who have not been 115

9 sensitized or exposed to the common dye precursors PPD and PTD. However, because a ME- PPD containing hair dye can still elicit reactions in at least 30% of the PPD-sensitized subjects, these individuals must be discouraged from using this hair dye. Concluding remarks People want to dye their hair. This carries some risk of becoming allergic. By gaining insight into the dose-response relationship of the classic hair dye precursor PPD we hope to have contributed to the process of risk management of hair dyes. 116

10 References 1. Cathelineau H. Note sur 1 cases d accidents provoques par teinture pour cheveaux a base de chlorhydrate de PPD. Bull Soc Franc Syph 19; 9: Brocq L. Les eruptions eczematiformes provoques pour une tinture pour chevaux a base de Chlorhydrate de Paraphenylendiamine. Bull Med 19; 12: Warbrick EV, Dearman RJ, Lea LJ et al. Local lymph node assay responses to paraphenylenediamine: intra- and inter-laboratory evaluations. J Appl Toxicol 1999; 19: Kligman AM. The identification of contact allergens by human assay. 3. The maximization test: a procedure for screening and rating contact sensitizers. J Invest Dermatol 1966; 47: de Groot AC. Side-effects of henna and semi-permanent black henna tattoos: a full review. Contact Dermatitis 2013; 69: Almeida PJ, Borrego L, Pulido-Melian E et al. Quantification of p-phenylenediamine and 2-hydroxy- 1,4-naphthoquinone in henna tattoos. Contact Dermatitis 2012; 66: El-Shaer NS, Badr JM, Aboul-Ela MA et al. Determination of lawsone in henna powders by high performance thin layer chromatography. J Sep Sci 2007; 30: Kind F, Scherer K, Bircher AJ. Contact dermatitis to para-phenylenediamine in hair dye following sensitization to black henna tattoos - an ongoing problem. J Dtsch Dermatol Ges 2012; 10: Council Directive of 27 July on the approximation of the laws of the Member States relating to cosmetic products (76/76/EEC). Off J Eur Union 1976: L262: U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition Office of Cosmetics and Colors Fact Sheet April 1, 2001, Updated September 1, Available at: gov/fdsys/pkg/uscode title21/pdf/USCODE-2010-title21- chap9-subchapvi-sec361.pdf. 11. European Commission, Scientific Committee on Consumer Products. Opinion on p-phenylenediamine, Available at: Thyssen JP, Andersen KE, Bruze M et al. p-phenylenediamine sensitization is more prevalent in central and southern European patch test centres than in Scandinavian: results from a multicentre study. Contact Dermatitis 2009; 60: Hillen U, Jappe U, Frosch PJ et al. Late reactions to the patch-test preparations paraphenylenediamine and epoxy resin: a prospective multicentre investigation of the German Contact Dermatitis Research Group. Br J Dermatol 2006; 154: Schnuch A, Lessmann H, Frosch PJ et al. para-phenylenediamine: the profile of an important allergen. Results of the IVDK. Br J Dermatol 200; 159: Xie Z, Hayakawa R, Sugiura M et al. Experimental study on skin sensitization potencies and crossreactivities of hair-dye-related chemicals in guinea pigs. Contact Dermatitis 2000; 42: Goon AT, Gilmour NJ, Basketter DA et al. High frequency of simultaneous sensitivity to Disperse Orange 3 in patients with positive patch tests to para-phenylenediamine. Contact Dermatitis 2003; 4:

11 Seidenari S, Mantovani L, Manzini BM et al. Cross-sensitizations between azo dyes and para-amino compound. A study of 236 azo-dye-sensitive subjects. Contact Dermatitis 1997; 36: Turchin I, Moreau L, Warshaw E et al. Cross-reactions among parabens, para-phenylenediamine, and benzocaine: a retrospective analysis of patch testing. Dermatitis 2006; 17: Uter W, Geier J, Lessmann H et al. Contact allergy to Disperse Blue 106 and Disperse Blue 124 in German and Austrian patients, 1995 to Contact Dermatitis 2001; 44: Thomas BR, White IR, McFadden JP et al. Positive relationship - intensity of response to p-phenylenediamine on patch testing and cross-reactions with related allergens. Contact Dermatitis 2014; 71: Devos SA, Van Der Valk PG. The risk of active sensitization to PPD. Contact Dermatitis 2001; 44: Dawe SA, White IR, Rycroft RJ et al. Active sensitization to para-phenylenediamine and its relevance: a 10-year review. Contact Dermatitis 2004; 51: Aalto-Korte K, Alanko K, Kuuliala O et al. Late reactions in patch tests: a 4-year review from a clinic of occupational dermatology. Contact Dermatitis 2007; 56: 1-6. Lachapelle J, Maibach H. Patch Testing, Prick Testing: A Practical Guide. Berlin, Heidelberg, New York, Springer-Verlag Isaksson M, Bruze M. Late patch-test reactions to budesonide need not be a sign of sensitization induced by the test procedure. Am J Contact Dermat 2003; 14: Higgins E, Collins P. The relevance of 7-day patch test reading. Dermatitis 2013; 24: Gawkrodger DJ, Paul L. Late patch test reactions: delayed immune response appears to be more common than active sensitization. Contact Dermatitis 200; 59: Diepgen TL, Coenraads PJ, Wilkinson M et al. Para-phenylendiamine (PPD) 1% pet. is an important allergen in the standard series. Contact Dermatitis 2005; 53: 15. Geier J, Ballmer-Weber BK, Dickel H et al. Monitoring contact sensitization to p-phenylenediamine (PPD) by patch testing with PPD 0.3% in petrolatum. Contact Dermatitis 2013; 69: Brasch J, Henseler T. The reaction index: a parameter to assess the quality of patch test preparations. Contact Dermatitis 1992; 27: Geier J, Uter W, Lessmann H et al. The positivity ratio--another parameter to assess the diagnostic quality of a patch test preparation. Contact Dermatitis 2003; 4: Sosted H, Menne T, Johansen JD. Patch test dose-response study of p-phenylenediamine: thresholds and anatomical regional differences. Contact Dermatitis 2006; 54: Ho SG, Basketter DA, Jefferies D et al. Analysis of para-phenylenediamine allergic patients in relation to strength of patch test reaction. Br J Dermatol 2005; 153: Goebel C, Coenraads PJ, Rothe H et al. Elicitation of the immune response to p-phenylenediamine in allergic patients: the role of dose and exposure time. Br J Dermatol 2010; 163: Karlberg AT, Shao LP, Nilsson U et al. Hydroperoxides in oxidized d-limonene identified as potent contact allergens. Arch Dermatol Res 1994; 26:

12 Rudback J, Hagvall L, Borje A et al. Characterization of skin sensitizers from autoxidized citronellol - impact of the terpene structure on the autoxidation process. Contact Dermatitis 2014; 70: Skold M, Borje A, Harambasic E et al. Contact allergens formed on air exposure of linalool. Identification and quantification of primary and secondary oxidation products and the effect on skin sensitization. Chem Res Toxicol 2004; 17: Hagvall L, Backtorp C, Svensson S et al. Fragrance compound geraniol forms contact allergens on air exposure. Identification and quantification of oxidation products and effect on skin sensitization. Chem Res Toxicol 2007; 20: Gerberick GF, Troutman JA, Foertsch LM et al. Investigation of peptide reactivity of pro-hapten skin sensitizers using a peroxidase-peroxide oxidation system. Toxicol Sci 2009; 112: Aeby P, Sieber T, Beck H et al. Skin sensitization to p-phenylenediamine: the diverging roles of oxidation and N-acetylation for dendritic cell activation and the immune response. J Invest Dermatol 2009; 129: Pot LM, Scheitza SM, Coenraads PJ et al. Penetration and haptenation of p-phenylenediamine. Contact Dermatitis 2013; 6: Divkovic M, Pease CK, Gerberick GF et al. Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization. Contact Dermatitis 2005; 53: Natsch A, Emter R, Ellis G. Filling the concept with data: integrating data from different in vitro and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing. Toxicol Sci 2009; 107: Schaefer H, Redelmeier TE, Lademann J. Skin penetration. In: Contact Dermatitis, 5th edition, Johansen JD, Frosch PJ, Lepoittevin JP (eds): Berlin, Heidelberg, Springer-Verlag, 2011: pp Gazith J, Schalla W, Bauer E et al. -Methoxypsoralen (-MOP) in human skin: penetration kinetics. J Invest Dermatol 197; 71: Kawakubo Y, Merk HF, Masaoudi TA et al. N-Acetylation of paraphenylenediamine in human skin and keratinocytes. J Pharmacol Exp Ther 2000; 292: Bonifas J, Scheitza S, Clemens J et al. Characterization of N-acetyltransferase 1 activity in human keratinocytes and modulation by para-phenylenediamine. J Pharmacol Exp Ther 2010; 334: Egawa M, Hirao T, Takahashi M. In vivo estimation of stratum corneum thickness from water concentration profiles obtained with Raman spectroscopy. Acta Derm Venereol 2007; 7: 4-. Pot LM, Alizadeh BZ, Ahrenberg D et al. Comment on no major role for glutathione S-transferase gene polymorphisms in sensitization to para-phenylenediamine and other xenobiotics: a study of association and a meta-analysis : reply from authors. Br J Dermatol 2011; 165: Basketter DA, English J. Cross-reactions among hair dye allergens. Cutan Ocul Toxicol 2009; 2: Blomeke B, Pietzsch T, Merk HF. Elicitation response characteristics to mono- and to N,N -diacetylpara-phenylenediamine. Contact Dermatitis 200; 5:

13 Blomeke B, Brans R, Coenraads PJ et al. Para-phenylenediamine and allergic sensitization: risk modification by N-acetyltransferase 1 and 2 genotypes. Br J Dermatol 2009; 161: Blomeke B, Brans R, Dickel H et al. Association between TNFA-30 G/A polymorphism and sensitization to para-phenylenediamine: a case-control study. Allergy 2009; 64: Brans R, Dickel H, Bruckner T et al. MnSOD polymorphisms in sensitized patients with delayed-type hypersensitivity reactions to the chemical allergen para-phenylene diamine: a case-control study. Toxicology 2005; 212: McFadden JP, Wakelin SH, Holloway DB et al. The effect of patch duration on the elicitation of paraphenylenediamine contact allergy. Contact Dermatitis 199; 39: Hextall JM, Alagaratnam NJ, Glendinning AK et al. Dose-time relationships for elicitation of contact allergy to para-phenylenediamine. Contact Dermatitis 2002; 47: Fischer LA, Johansen JD, Menne T. Nickel allergy: relationship between patch test and repeated open application test thresholds. Br J Dermatol 2007; 157: Lundov MD, Zachariae C, Johansen JD. Methylisothiazolinone contact allergy and dose-response relationships. Contact Dermatitis 2011; 64: Andersen KE, Johansen JD, Bruze M et al. The time-dose-response relationship for elicitation of contact dermatitis in isoeugenol allergic individuals. Toxicol Appl Pharmacol 2001; 170: Fischer LA, Menne T, Voelund A et al. Can exposure limitations for well-known contact allergens be simplified? An analysis of dose-response patch test data. Contact Dermatitis 2011; 64: Goebel C, Diepgen TL, Krasteva M et al. Quantitative risk assessment for skin sensitisation: Consideration of a simplified approach for hair dye ingredients. Regul Toxicol Pharmacol 2012; Sosted H, Rustemeyer T, Goncalo M et al. Contact allergy to common ingredients in hair dyes. Contact Dermatitis 2013; 69: European Commission, Scientific Committee on Consumer Safety. Opinion on Hydroxyethyl-p-phenylenediamine sulfate, Available at: committees/consumer_safety/docs/sccs_o_017.pdf.

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