Cross‐elicitation responses to 2‐methoxymethyl‐p‐phenylenediamine in p‐phenylenediamine‐allergic individuals: Results from open use testing and diagnostic patch testing

Background Allergic contact dermatitis caused by p‐phenylenediamine (PPD) is a health concern for hair dye users. Because of its lower sensitization potency, the PPD derivative 2‐methoxymethyl‐p‐phenylenediamine (ME‐PPD) has been developed as an alternative hair dye for primary prevention. However, cross‐elicitation responses can occur in PPD‐allergic subjects. Objectives To compare cross‐elicitation responses to ME‐PPD in open use and diagnostic patch testing of PPD‐allergic subjects with hair dye‐related allergic contact dermatitis. Methods Reactions to ME‐PPD were investigated in 25 PPD‐allergic subjects by performing (1) 45‐minute open use testing with a hair dye containing 2.0% of either ME‐PPD or PPD, and (2) patch testing with increasing ME‐PPD concentrations (0.1%–2.0% pet.). Results Of the 25 PPD‐allergic subjects, 21 (84%) reacted to open use testing with a hair dye containing 2.0% PPD, and testing with 2.0% ME‐PPD led to cross‐elicitation in 12 (48%). When patch tested with increasing ME‐PPD concentrations, 13 (52%) cross‐reacted at 0.1% (lowest dose) and 21 (84%) at 2.0% (highest dose), indicating decreased reactivity as compared with published PPD dose‐response data. Conclusion In line with the decreased cross‐reactivity of ME‐PPD in hair dye open use testing, PPD‐allergic subjects show an attenuated cross‐elicitation dose response to ME‐PPD in patch testing.

methoxymethyl side-chain into PPD, resulting in a hair-dyeing performance equivalent to that of PPD and TDA. Analysis of the skin sensitization potency of ME-PPD in in vitro studies indicated a lower skin sensitization potency than that of PPD and TDA. In vivo, the LLNA showed a moderate skin sensitization potency of ME-PPD. Therefore, induction of skin sensitization has been considered to be unlikely when ME-PPD is used to replace PPD or TDA in hair dyes. 3 Accordingly, ME-PPD has been developed for the prevention of skin sensitization, and not for individuals who have already been sensitized to other hair dye precursors, such as PPD and TDA. However, it is known that many individuals who are allergic to PPD or TDA continue to dye their hair, and they may use hair dye products that contain ME-PPD instead of PPD or TDA. 4 Therefore, the current study investigated cross-elicitation responses to an ME-PPD-containing hair dye under open use test conditions in PPD-allergic individuals with a history of hair dye-related allergic contact dermatitis. Furthermore, their crosselicitation dose response to ME-PPD was determined under diagnostic patch test conditions, and compared with PPD elicitation dose-response data previously published by Søsted et al. 5

| METHODS
Twenty-five adult individuals with a previously documented positive patch test reaction to PPD 90 μg/cm 2 (TRUE Test; SmartPractice Europe, Reinbek, Germany) or PPD 1% pet. (Chemotechnique Diagnostics, Vellinge, Sweden) and a history of allergic contact dermatitis caused by hair dye were included. The exclusion criteria were as follows: skin anomalies or active dermatitis on the volar aspects of the forearms or on the back, and the use of immunosuppressive medication (including, but not limited to, oral corticosteroids, cyclosporine, azathioprine, and methotrexate) during the 4 weeks prior to inclusion. All tests were performed at the Department of Dermatology, University Medical Center Groningen, The Netherlands, and assessed according to ESCD guidelines on day (D) 2, D3, and D7. 6 Of the 25 subjects included in this present study, 8 had participated in a previous ME-PPD open use test study. 7 In the previous study, 6 of these 8 reacted positively to ME-PPD under hair dye conditions (open use test), and the other 2 did not. The study was approved by the local ethics committee of the University Medical Center Groningen.

| Open use testing
Open use tests were performed on the volar aspects of both forearms.
The patch test preparations, the vehicle (Koleston Perfect formula without fragrance) containing the hair dye precursors (PPD 4.0% or ME-PPD 4.0%, free base) and couplers (1.9% 2-methyl-5-hydroxyethylaminophenol; 3.6% 2-methylresorcinol) and the hydrogen peroxide-based developer solution (6.0% wt/wt Welloxon) were provided by Procter & Gamble Service (now represented by Coty, Darmstadt, Germany). The couplers were selected on the basis of their negligible sensitization potency as determined with the LLNA, each with an EC3 greater than 50. 8,9 The hair dye test product was always freshly prepared by mixing the tint (containing PPD or ME-PPD, and the couplers) with the hydrogen peroxide solution by use of a small wooden stick (1:1, 90 μL each), resulting in solutions containing 2.0% PPD and 2.0% ME-PPD, respectively. A dye-free test product, also mixed with hydrogen peroxide as described above, was used as a negative control. A 100-μL aliquot of the final PPD-containing or ME-PPD-containing product was applied directly to the volar forearm with a micropipette in a 3.8-cm 2 area marked by a round adhesive tape with a diameter of 22 mm. PPD was tested on the volar aspect of 1 arm, and ME-PPD and the negative control were tested on the other, so that an extreme positive reaction to PPD would have no influence on a possible elicitation response to ME-PPD. The contours of the round adhesive tape were marked with a Chemo Skin Marker (Chemotechnique Diagnostics), in order to enable recognition of the test site at follow-up. The test areas were rinsed off with water after 45 minutes of application to simulate hair-dyeing conditions.

| Diagnostic patch testing
Patch testing was performed on the back with ME-PPD 0.1%, 0.25%, 0.5%, 1.0% and 2.0% pet. in Van der Bend chambers (Van der Bend, Brielle, The Netherlands), fixed with Fixomull Stretch (BSN Medical, Hamburg, Germany). In the first 10 subjects, ME-PPD was only patch tested in concentrations of 0.5%, 1.0% and 2.0% pet., after which the protocol was adapted to also include patch testing with ME-PPD 0.1% and 0.25% pet. The first 10 subjects were recalled for testing with these 2 concentrations, and all except for 2 subjects were additionally tested.

| Evaluation and statistics
Responses to the open use test were recorded at 60 minutes after application (15 minutes after rinsing of the area), and, together with the patch test readings, on D2, D3, and D7, and graded according to ESCD guidelines. 6 For data analysis, the maximum open use test/ patch test reactions were aggregated as the outcome.
Results are presented as the proportion of PPD-allergic patients who responded with a cross-elicitation response to ME-PPD as indicated by a positive patch test reaction for each dose. Binary logistic regression was performed, and a dose-response curve was plotted in order to investigate the threshold dose for cross-elicitation for different proportions of the study population. The probability of a positive response P(x), where x represents the natural logarithm of the dose, for a given dose is as follows: The effective dose (EDy), that is, the hypothetical dose at which a   Table 2.
Twelve of the 25 subjects (48%, 95%CI: 28%-68%) showed crosselicitation reactions to the open use test with hair dye containing 2.0% ME-PPD (Table 2) 7 None of these 8 subjects had been exposed to ME-PPD hair dye in any other manner prior to participation in the present study.
The 2 who did not react to the ME-PPD open use test in the previous study did not react to ME-PPD in the present study, suggesting that they were not actively sensitized in the previous study.
3.2 | Dose-dependent cross-elicitation reactions to ME-PPD in diagnostic patch testing  Table 2). No irritant patch test reactions were seen.  Open use hair dye ME-PPD 2% ME-PPD 0.1% ME-PPD 0.25% ME-PPD 0.5% ME-PPD 1.0% ME-PPD 2.0% Open use testing with 2.0% ME-PPD in a hair dye showed crosselicitation in 12 of the 25 PPD-allergic subjects, that is, in 48% ( Figure 1A). In contrast, open use testing with 2.0% PPD ( Figure 1B) showed an elicitation response in 84% (21/25). Furthermore, the reaction strength of the cross-elicitation response was reduced in 11 of the 12 (92%) PPD-allergic subjects who reacted to both PPD and ME-PPD open use tests ( Table 2). This is in line with data from previous cross-elicitation studies, 7,10 indicating partial cross-reactivity to ME-PPD under hair dye use conditions. In those previous studies, the response was 30% with an exposure time of 30 minutes. The observed higher response of 48% to the 45-minute ME-PPD hair dye open use test in the present study confirms previous findings with PPD showing that an increase in the exposure time from 30 to 60 minutes increased the amount available for (cross-)elicitation by >2-fold. 11 We also investigated whether non-responsiveness to ME-PPD open use tests in PPD-allergic subjects is dependent on the limited 45-minute exposure time. Exposure to increasing ME-PPD patch test doses for 48 hours under occlusion showed a dose-dependent increase in the cross-elicitation response: at the lowest patch test dose of 0.1%, the response of 52% was similar to that with hair dye open use testing with 2.0% ME-PPD (48%), indicating that a comparable immune stimulus can be provided with either short high-dose exposure or longer low-dose exposure. This interpretation is further supported by similar cross-elicitation reaction strengths in 5 of the 9 subjects (55.0%) who showed elicitation reactions to both 0.1% patch tests and 2.0% open use tests. 12 With increasing patch test doses up to 2.0%, an increasing number of PPD-allergic subjects cross-reacted to ME-PPD, with 84% showing positive reactions at the maximum tested dose of 2.0% ( Figure 1). These findings indicate that the majority of PPD-allergic patients are probably able to cross-react to ME-PPD, provided that the dose is high enough and/or the exposure time is long enough. In line with the responses to open use testing ( Figure 1A), a higher percentage of the extreme PPD responders than of the weaker PPD responders cross-reacted at lower ME-PPD patch test doses. A possible explanation might be a higher number of PPD-specific memory T cells being present in the extreme PPD responders.
On comparison of the ME-PPD cross-elicitation patch test dose response obtained in the present study with the patch test dose response to PPD obtained in 15 PPD-allergic subjects, 5 the curve for ME-PPD is shifted further towards higher concentrations ( Figure 3). In other words, over the entire dose range, higher ME-PPD concentrations are needed to generate a response rate comparable to that seen with PPD. This is also indicated by the 4-fold increased ED50 value for ME-PPD vs PPD, and is in line with the hypothesis that ME-PPD is a less potent allergen. 3,5 The measured exposure level (MEL) (representing the concentration that is available to the skin) of the applied PPD patch dose of 1.0% for 48 hours is 200 μg/cm 2 . This is an order of magnitude higher than the MEL for PPD in hair dye open use conditions for 30 minutes, which ranges from 6.8 to 21 μg/cm 2 . 11 This PPD concentration was found to be sufficient to elicit a reaction in 84% of PPD-allergic patients. For ME-PPD, the MELs for 1.8% applied under hair dye use conditions were 8.75 and 10.21 μg/cm 2 for 30 minutes and 60 minutes, respectively, 3 and were thus similar to the MEL for PPD. Consequently, it is reasonable to assume that the MEL for patch test conditions with exposure to 2% ME-PPD is at least >10 fold higher and in line with the higher response observed in patch testing than in open use testing.
In agreement with the present results, a study from Kock et al investigating cross-elicitation reactions to repeated hair dyeing with ME-PPD in PPD-allergic and/or TDA-allergic individuals showed that 29 of 43 were able to tolerate repeated hair colouring with an average of 9 treatments per year. 13 In that study, 9 subjects did not react to the initial 45-minute open use test on the forearm, and 7 of  them developed allergic contact dermatitis symptoms during the first hair colouration with ME-PPD. Together with our findings, these data suggest that an individual threshold for cross-elicitation to ME-PPD exists in PPD-allergic subjects. We assume that the skin site-specific occurrence of the cross-elicitation responses to ME-PPD between forearm and back (in the present study) or scalp (in the study from Kock et al 13 ) is related to the presence of (sufficient) residual memory T cells. Similar findings have been reported for patients with allergy to nickel: a rapid response to nickel only occurred after re-exposure to nickel on the exact body site that had been previously exposed, and the magnitude of the elicitation responses correlated with local recruitment of epidermal CD8 + T cells. 14 In line with previous studies on ME-PPD, our hair dye open use and patch test data further support the recommendation for hair dyeallergic individuals to avoid hair colouring, because cross-elicitation responses to ME-PPD cannot be excluded. 7,10,13 Despite intensive investigations, the precise hapten responsible for PPD allergy has not been identified. 15 Considering that induction of sensitization with PPD has been shown to be dependent on duration of exposure, 16 PPD oxidation to protein reactive auto-oxidation products such as Bandrowski's base may be involved in elicitation. 17 Furthermore, PPD undergoes N-acetylation when entering the epidermis, and thus becomes deactivated. 18 In line with the findings for PPD, both oxidative activation of ME-PPD and deactivation by N-acetylation have been described in vitro, and human skin has been shown to actively Nacetylate ME-PPD. 3 Correspondingly, the structural similarity to PPD and the consistent activation and deactivation mechanisms suggest that, in PPD-allergic individuals, the concentration-dependent immune response to ME-PPD is based on the partial inability of T cells to differentiate between PPD-derived and ME-PPD-derived haptens. 10

| CONCLUSION
Cross-elicitation analysis in PPD-allergic individuals indicates that ME-PPD is a less potent allergen than PPD under simulated hair dye use conditions and under diagnostic patch test conditions. The cross-reactivity shows a clear dose dependency, with increasing cross-reactivity to ME-PPD being seen at higher patch test doses. ME-PPD can only be considered to be an alternative hair dye for primary prevention of sensitization.