Introduction of a methoxymethyl side chain into p-phenylenediamine attenuates its sensitizing potency and reduces the risk of allergy induction
Introduction
Oxidation hair dyeing with the p-amino aromatic compound p-phenylenediamine (PPD) has a long tradition (Corbett, 1999). This key primary intermediate is still used in many permanent hair dye products based on its excellent color performance. Following oxidation of the primary intermediate by hydrogen peroxide, color performance is dependent on the high degree of reactivity of its oxidized form with couplers to form dimeric and trimeric reaction products within a short period of time (Scientific Committee on Consumer Safety (SCCS, 2010b)).
Apart from the excellent hair coloring property PPD is well known as the most important allergen associated with hair dye-related allergic contact dermatitis reviewed in (Goebel et al., 2012, Krasteva et al., 2009). Historically, introduction of a side chain into the PPD molecule in the 2 position was studied to assess if the skin sensitizing properties can be modulated while keeping the coloring performance. Successful attempts regarding performance were the 2-methylderivative p-toluylenediamine (PTD) and to a lesser degree the 2-hydroxyethyl derivative hydroxyethyl-PPD (HE-PPD). However, no major reduction of the sensitizing potency was found: based on maximized potency data derived from skin sensitization tests such as the local lymph node assay (LLNA), PTD and PPD are both categorized as extreme sensitizers, while HE-PPD is categorized as strong sensitizer (SCCS, 2013a).
Advancements in understanding the mechanistic steps underlying skin sensitization revealed in the case of pre-haptens, such as PPD, that oxidation, epidermal bioavailability, protein reactivity, and dendritic cell (DC) activation are critical parameters (Aeby et al., 2009, Jenkinson et al., 2009, McFadden et al., 2011). In addition, aromatic amine hair dyes are known to undergo enzymatic N-acetylation in the skin (Goebel et al., 2009, Kawakubo et al., 2000, Nohynek et al., 2005) and N-acetylation of PPD was shown to prevent skin sensitization in the LLNA (Aeby et al., 2009).
Therefore, in the present paper we asked how introduction of a methoxymethyl side chain into PPD, resulting in 2-methoxymethyl-p-phenylenediamine (ME-PPD), impacts the skin sensitizing properties including skin metabolism.
For this purpose, we investigated a) the bioavailability and the metabolic fate using human skin ex vivo, b) the reactivity towards protein using the peptide reactivity assay in the presence and absence of an oxidizing system (Gerberick et al., 2009) and c) the ability to activate THP-1 cells, used as surrogate DCs, for the expression of the costimulatory molecule CD86 (Migdal et al., 2013, Yoshida et al., 2003).
In the LLNA we further assessed, how the interplay of the individual mechanistic steps analyzed is related to specific T lymphocyte recognition and proliferation, because the magnitude and vigor of the proliferative response induced determine the extent to which skin sensitization will be acquired (Kimber et al., 2011). Thus, we determined the skin sensitizing potency of ME-PPD by calculating the EC3 value (concentration necessary to induce a 3-fold increase above the vehicle control) representing the threshold for the induction of skin sensitization, referred to as the non expected skin sensitization induction level (NESIL).
Finally we applied a quantitative risk assessment (QRA) approach for hair dyes (Goebel et al., 2012) to determine if usage of ME-PPD in hair dye products may be associated with the risk to induce skin sensitization by comparing the skin sensitizing potency (NESIL) with the measured exposure level (MEL) of the skin following a typical hair coloring application.
Section snippets
Material and methods
Analytical standards of all mono and di-acetylated derivatives, parent compounds as well as the hair color formulations used for the ex-vivo skin studies were from the Procter and Gamble Service GmbH, Darmstadt, Germany. All other chemicals were of the highest grade from Sigma Aldrich. Radioactive ME-PPD [ring-U-14C]ME-PPD-sulfate with a specific activity of 20 mCi·mmol-1 was used (GE-Healthcare, Cardiff, UK).
Determination of skin exposure and metabolic fate of ME-PPD
An experimental design with frozen pig skin samples was used to determine the relevant actual skin exposure to ME-PPD following standard use conditions of oxidative hair dyeing referred to as the measured exposure level (MEL) in the context of skin sensitization, i.e., the ME-PPD amounts remaining on/in the skin surface (i.e. Stratum corneum) following the rinsing step and the bioavailable amounts (i.e. viable skin and receptor fluid) (Goebel et al., 2010). A MEL of 8.75 μg/cm2 was found when we
Discussion
In the present paper, we asked how modifying the side chain structure of PPD impacted the skin sensitizing properties of the resulting 2-methoxymethyl-p-phenylenediamine (ME-PPD). For this purpose, we characterized how ME-PPD passes through the key stages of the innate immune response, namely skin exposure (MEL), epidermal bioavailability and metabolism, protein reactivity, and dendritic cell activation in comparison to the commonly used hair dye primary intermediates PPD and PTD as benchmarks.
Conflict of interest
Carsten Goebel, Helga Rothe, John Troutman, Harald Schlatter and Frank Gerberick are employees of a company selling cosmetic products including hair colorants. The hair dye ingredients studied in this paper are currently used in commercial products marketed by this company. Brunhilde Blömeke and Jenny Hennen participated as study directors and experts in skin sensitization. The authors alone are responsible for the content and writing of the paper. The manuscript is the joint work of the
Acknowledgments
The authors would like to thank Pia König for expert technical support.
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