Phenylenediamine (PPD) Concentrations after Hair Dye Mixing: A Call for Safety Reassessment

Study of P-Phenylenediamine (PPD) Concentrations after Hair Dye Mixing: A Call for Safety Reassessment


Para-phenylenediamine (PPD) is a chemical that is widely used in hair dyes. Multiple safety and regulatory agencies have categorized PPD as a potent sensitizer. In addition, PPD has carcinogenicity and genotoxicity attributes and, consequently, it is regulated at a maximal concentration of 2.0%. The aim of this study was to test whether the limit for PPD is surplus, and hence whether the consumer may be exposed to unnecessarily PPD levels. Experimentally, the analysis of PPD was performed using HPLC, where method validation and an inter-laboratory comparison test (ILC) were conducted to evaluate method performance. Thirty-three commercial products were analyzed, and five products were chosen to study the unconsumed PPD. Successfully, the implemented method confirmed its suitability and validity for the determination of PPD. For ILC results, PPD levels were 0.97 ± 0.04% and 0.92 ± 0.02%, quantified by our laboratory and an accredited laboratory, respectively. For all products, the initial concentration (T0) of PPD was lower than the regulatory limit. After 45 min, the content of PPD significantly reduced compared to T0. One product showed unconsumed PPD to be as high as 96% following the recommended dyeing time. In conclusion, the existence of high levels of unreacted PPD increases the likelihood of allergic events and elevates the risk of PPD-related chemicals. Collaborative efforts between industries, regulatory bodies, and health-related decision makers are deemed necessary to establish safe concentrations for PPD.


Hair is a skin derivative representing definite characteristics of the human body. Structurally, the root and the shaft are the two main components of human body hair. Specifically, the shaft elements are organized into three layers: (1) the cuticle, which is the outermost protective part consisting of keratinized cells; (2) the cortex, which is the middle part forming the main bulk and containing the natural hair color pigments; (3) and medulla, which is the innermost layer [1,2]. Several factors can contribute to relative changes in hair texture or color, including diet, genes, environment, and hair dyes [3].
Hair dye products have become available in markets for various cosmetic purposes, such as covering gray hair, substituting hair color, and potentiating its natural color retention and longevity. These factors have accelerated a high demand for hair color products, concomitant with population aging [4,5]. As a result, the hair dye market has developed over the years into a multibillion-dollar industry, with permanent hair dyes representing the majority of its income (ca 70%). The popularity of permanent hair dyes arises from their desirable long-term effects and ease of application [5].
Hair dyes are divided into the following categories according to their origin: (1) vegetable hair dyes of which henna is the most widely used; (2) mineral hair dyes (silver nitrate or lead salts) requiring progressive usage and color treatments to reach the desired color lasting for weeks; and (3) synthetic hair dyes. Synthetic hair dyes are further classified, based on their permanence degree, into four types. (1) Temporary hair dyes, which last for a few days due to their high molecular weight and deposition on the cuticle surface. (2) Semi-permanent hair dyes that last for weeks due to their low molecular weight and shallow cortical penetration. (3) Permanent dyes, for which the coloring persists permanently due to their low molecular weight and deep cortical penetration. (4) Hair-bleaching dyes that oxidize melanin in the hair cortex in order to lighten the shade of the natural hair prior to hair coloring [6,7].
Mechanistically, permanent hair dyes normally constitute non-colored active ingredients that are subsequently oxidized to form the desired color. Accordingly, the term oxidative hair dye emerged [1,6]. There are two major components within oxidative hair dye formulations, i.e., the precursor part and coupler part. When mixed with hydrogen peroxide (developer), transient quinonediimine intermediates are formed. Consequently, these chemicals react with the coupler agent to generate the desired hair dye molecule. Of note, the entire dyeing procedure necessitates an oxidizing agent (frequently hydrogen peroxide) and an alkaline medium to ensure wide penetration of the staining constituents to the cuticle. Therefore, the oxidative dye products are originally packed into two separate bottles. One bottle contains the precursor and the couplers, whereas the other contains the developer [4,6].
During the past fifty years, p-phenylenediamine (PPD) has frequently been used as a primary precursor in the process of manufacturing oxidative hair dye products. PPD has the ability to provide a wide range of colors, hence gaining popularity among manufacturers [8]. Despite that, the safety of PPD is questionable as many health risks are associated with the use of PPD in both short and long-term applications. For example, there is accumulating evidence that PPD is associated with multiple cases of dermatitis and allergies [9,10,11,12,13,14]. In addition, PPD is considered a potent skin sensitizer, and thus a patch test is recommended before using any hair dyes containing PPD.
Furthermore, PPD may cause cross-allergic reactions to other chemicals [15]. Importantly, several experiments have shown a significant association between the use of products containing PPD and mutagenicity, according to the Scientific Committee on Consumer Products (SCCP) [10]. Owing to PPD risks, some countries such as Germany, France, and Sweden have banned the use of PPD during the twentieth century [11,12,13]. Alternatively, other regulatory agencies have set stringent levels for PPD in cosmetic products. For example, the European Union Cosmetic Directive Regulation has banned PPD in topical products that are intended for superficial purposes while allowing PPD inclusion in cosmetic products marketed as oxidizing coloring agents with a maximum concentration of 4% (free base). Specifically, the permitted concentration is 2% (free base) when mixing PPD with hydrogen peroxide following the preparation protocol of hair dyeing products [4,5,6]. On the contrary, several research studies have demonstrated violations of these limits. For example, a report showed that PPD was detected in 76 out of 115 products with a concentration range of 2.2 to 3.4%, rendering these products incompliant [11].
The well-documented evidence of PPD-related risk in cosmetics products raises alarm about the safety of this chemical. Recent studies have focused on designing new PPD alternatives to evade the health concerns associated with PPD usage [16,17]. A safe limit for PPD in hair dye products that can provide desirable coloring with minimum body exposure to PPD needs to be carefully defined. In this study, we aimed to test whether the current limit for PPD in hair dye products (i.e., 2%) is beyond a sufficient coloring need by measuring the PPD after mixing the ingredients of commercial color dyes. Using a validated HPLC method, we measured the unreacted amount of PPD at different time intervals to evaluate the potential of the current PPD limit to be overestimated. The output of the current study can potentially contribute to the regulatory science about the limits and regulations regarding PPD in cosmetics in general and, in particular, hair dyes.


The risk associated with the use of PPD has motivated scientists to develop multiple analytical techniques to detect and quantify this chemical in cosmetic products to ensure their safety. Furthermore, because hair dyeing is based on oxidative chemical reactions, one can expect a variety of chemicals and intermediates with structural similarities, making their detection cumbersome. Consequently, several chromatographic methods have been reported for the analysis of the intermediates of oxidative hair dyes [18,22,23,24]. Some of these methods may be suitable only for checking the purity of the raw materials, while others may also be used for the analysis of intermediates of hair dyes in cosmetic formulations. ICP/MS, HPLC/MS, and GC-MS are reported analytical methods used to separate and determine the hair dye’s PPD intermediates [4]. However, these methods are costly, time-consuming, and some of them involve complicated procedures for sample preparation, such as extraction and chemical derivatization [22]. Because of that, a convenient and relatively easy-to-use technique is demanded in order to be used for routine analysis, and hence the advantage for the HPLC technique becomes visible. Practically, reported HPLC methods for the analysis of PPD in hair dye products suffer from major problems related to the quality of the chromatographic peak. Severe tailing, asymmetric chromatographic peak, and the inherent instability of PPD in hair dyes are common issues associated with HPLC analysis for any PPD-contained products. Fortunately, reversed-phase ion-pairing liquid chromatography has proven to counteract those issues with chromatographic separation [24]. In the current study, we used the ion-pairing technique in which we adapted the previous method with minor modifications. In order to improve peak shape, enhance detection sensitivity, and obtain a high response, a specified amount of ion-pair reagent was needed. We used 1-heptane sulfonic acid sodium salt as an ion-pair reagent to minimize the peak tailing. During method optimization, the resolution between the oxidizing hair dye components of commercial products was challenging. The original method used a flow rate of 1.0 mL/min, and authors achieved satisfactory resolution between some oxidizing components, but not for others [18]. In order to overcome this problem, authors recommended decreasing the flow rate from 1.0 to 0.8 mL/min, and this is the strategy we used in the current study.


A method for PPD quantification was successfully implemented and validated, circumventing the severe tailing and asymmetric chromatographic peak of PPD by using reversed-phase ion-pairing liquid chromatography. All tested samples complied with the current regulatory limits of PPD. The analysis of unreacted PPD revealed as much as 77% prevalence following extended dyeing time longer than what is recommended by the manufacturer. Eventually, higher PPD levels would likely be observed after strictly applying the manufacturers’ recommended dyeing times. Keeping in mind the health risks associated with PPD, as well as the carcinogenic potential of PPD-related chemicals, unconsumed PPD needs to be further explored and evaluated by regulatory bodies. Ideally, consumers are expected to be exposed to minimum levels of hazardous chemicals in hair dyes while obtaining the desired hair color. Collaborative efforts between industry, regulatory bodies, and health-related decision makers are deemed necessary to establish safe concentrations of sensitizing chemicals in hair dyes, particularly PPD.