Ingredients, Chemical Analysis and Safety of Marketed Tattoo Ink Stock Products: Photostability and Breakdown Products of Pigments Currently Used in Tattoo Inks

Organic pigments are the main components of coloured tattoo inks. Appropriate toxicological data being unavailable, the legal restrictions for tattoo inks in Europe still lean on restrictions for cosmetics, toys and textiles [1]. Specific toxicological data are desperately needed to establish a list of pigments safe for tattooing. From today's perspective, however, it is unlikely that such data will be available in the near future. At the moment, it seems more promising to improve consumer safety using chemical analysis to sort out those pigments which potentially can degrade to allergens or carcinogenic, mutagenic or reprotoxic compounds in the human body.

Tattoos fade with time. Several mechanisms are thought to play a part in this. Some fading can be attributed to the transport of the injected pigments away from the skin into the regional lymph nodes and other parts of the body [2, 3] where their fate remains unknown. Pigments may also be metabolised by enzymes [4,] as was discussed for azo reductases, which are able to split azo colourants into hazardous aromatic amines [5]. If this also happens in the dermis, where metabolic activity is low [6,] seems questionable. Pigments, however, are transported to other parts of the body where they might be metabolised.

As pigments remain in the skin for the rest of life, lightfastness is an important criterion for them. Colourants used for cars and plastic materials for outdoor purposes must withstand solar radiation and have therefore been deemed suitable for tattoo inks, regardless of the possible toxicological consequences. While their lightfastness is tested under outdoor conditions, screening for photodegradation products is not performed.

If people get tired of their tattoos, removal by laser irradiation is the method of choice. Upon absorbing laser light energy, pigment crystals are superheated to several hundred degrees Celsius and break into fragments which are washed away by lymphatic transport [7]. Thermic decomposition of the pigments is also to be expected.

Up to now, only a few studies have investigated the photodegradation of pigments used in tattoos. C.I. 11741, a yellow mono-azo pigment, was subjected to simulated solar light in a solution of tetrahydrofuran [8]. Three of the major fragments (table 1) were identified, suggesting cleavage at the hydrazine and amide groups. Photodegradation was also studied for the red mono-azo pigment C.I. 12315, which was dissolved in organic solvents and subjected to ultraviolet B (UVB) radiation (2.5-8 h; 14-43 J/cm²) and natural sunlight (10-110 days) [9]. Depending on the solvent and light source, a slight discolouration up to the complete mineralisation of C.I. 12315 was observed. The nature of degradation products (table 1) suggests a reductive cleavage of the azo bridge as well as cleavage at the site of the azo bond under loss of nitrogen. The fate of this pigment was also studied in vivo using tattooed mice as a model [10]. Irradiation with simulated solar radiation for 32 days caused a pigment reduction of about 60% in the skin. The decomposition products identified in vitro, however, were not observed in vivo. The authors reckoned that these substances were formed temporarily but disappeared either by on-site metabolism, additional photochemistry or dissemination throughout the body. This would not be surprising, as the expected decomposition products are much more soluble than the pigment itself. While the in vitro studies investigated pigments dissolved in strong solvents, in the dermis, pigments occur as little crystals that are mostly surrounded by body fluids [7]. We therefore designed a study [11], where ready-to-use tattoo inks containing often-used pigments [12] were suspended in water or squeezed between glass plates as a thin layer. The samples were then exposed to natural sunlight for at least 35 days in late spring, with the UVB part of the spectrum being partly blocked by the glass plates. Liquid chromatography with ultraviolet/visible (UV/VIS) and mass spectrometric detection were used as methods. The irradiated samples were also screened for bioactive substances using thin layer chromatography followed by Vibrio fisheridetection. Irradiation between glass plates resulted in a stronger photodegradation. With the exception of a violet ink, all exposed samples produced bioactive fragments. Examination of the data revealed that for the pigments C.I. 51319, C.I. 73915, C.I. 74160 and C.I. 74260, no bioactive substances were detected. A highly bioactive substance that was present in all samples containing the pigments C.I. 21095, C.I. 21110 and C.I. 21115 was identified to be 3,3′-dichlorobenzidine (3,3′-DCB), a structural element of all these pigments that was released by reductive cleavage of the azo bonds. A list of several other identified degradation products is shown in table 1. In a second study [13,] pure pigments were submitted to the glass-plate experiment. In general, irradiation by artificial sunlight, which is commonly used for determining the photostability of sunscreens, gave similar results as irradiation with natural sunlight. Comparison of the degradation of pigments in inks with that of the pure pigments showed a much faster reaction in the inks. We assume that the finer pigment particles in tattoo inks have a higher specific surface than the agglomerated particles of pure pigments, thus leading to higher reaction rates. In most cases, degradation of inks led to the same compounds as were found in irradiated pigments. Especially, azo pigments in tattoo inks were easily degraded under artificial sunlight, whereas other important pigments, like quinacridones (C.I. 73915, 73907), phthalocyanins (C.I. 74160, 74260, 74265) or dioxazines (C.I. 51319, 51345), seemed fairly stable. Chromatographic runs revealed many peaks but only a few of them were identified (table 1). The main problem with in vitro assays lies in the elimination of photolabile products due to further reactions when under continued irradiation. In vivo, soluble ink components and degradation products are washed away by body fluids. In an attempt to preserve these photolabile degradation products, artificial irradiation with only the visible part of the spectrum was used, thus as a further benefit, simulating the situation of pigments in the dermis where only UVA and visible radiation can penetrate; UVB irradiation being mostly blocked by the epidermis. Figure 1 shows aliquots of orange tattoo ink, containing C.I. 21110 and titanium dioxide, that were subjected to 7 days of either artificial sun irradiation or visible light irradiation [13]. Exposure to visible light led to an obvious degradation of the inks. Full-spectrum light not only promoted degradation but also led to a variety of new degradation products when compared to aliquots exposed only to the visible spectrum. Interestingly, the expected degradation product 3,3′-DCB was only found in the full-spectrum-irradiated sample, whereas on the other hand 3,3′-dichlorobiphenyl (PCB-11) was only found in the VIS-irradiated sample.

In the first study investigating breakdown products after laser irradiation [14], the authors chose two red mono-azo pigments (C.I. 12315 and C.I. 12460) because red tattoos are the cause of most allergic reactions to tattooing. Laser irradiation performed on suspensions of the pigments in acetonitrile resulted in several fragments, of which three were identified (table 1). In the case of C.I. 12315, two of the identified products (4-nitrotoluene and 2-amino-4-nitrotoluene) were also found after irradiation with sunlight. In a second study [13], commercial tattoo inks instead of pure pigments were diluted with water and irradiated as in the first study [14]. For the azo pigments C.I. 11767, C.I. 12370, C.I. 21095, C.I. 21108 and C.I. 21110 similar degradation products as those observed in the first study [14] were detected. In the case of C.I. 11741, the expected reduction of the azo bridge could not be confirmed due to the presence of o-anisidine as an impurity in high concentration levels. The results of the tests are summarised in table 1.

Identifying the photodegradation products of pigments is a difficult task. Even for pure pigments only few studies are available, and all have downsides. Photodegradation of C.I. 12315 was shown in vivo with mice [10,] but the postulated decomposition products were not detected, presumably because they were eliminated during the period of irradiation. In vitro tests, besides their obvious shortcomings, have another flaw: In contrast to the in vivo situation, where decomposition products are constantly washed away from the dermis, they remain at their site of reaction and are prone to decay and react with other fragments. Subsequent screening would then reveal fragments unrelated to in vivo conditions. More lightfast pigments take longer to degrade, making it more difficult to detect photolabile degradation products. Some samples can even be completely mineralised under prolonged irradiation. Several potential azo pigment degradation products, like naphthol AS-type compounds or acetoacetarylamides (e.g., o-acetoacetanisidide), are not only formed after irradiation [13], they also occur as impurities in pigments, thus complicating the interpretation of irradiation experiments. Upon first view, inks do not appear suitable for in vitro experiments. Their ingredients, e.g. surfactants, thickeners or preservatives, form a reaction environment which is not relevant in vivo. Still, the investigation of inks has certain advantages. Presumably because of their very fine pigment dispersions, photodegradation is accelerated, and degradation products may be easier to detect. Furthermore, effects on photostability by the specific pigment combination used have to be taken into account. When new inks containing C.I. 21110 were irradiated [13], the previously found degradation product 3,3′-DCB [11] was only found in traces. We suppose that the much higher content of titanium dioxide present in the older sample had a catalysing effect on C.I. 21110 degradation. Titanium dioxide is very often used in tattoo inks.

Laser irradiation experiments seem to better simulate in vivo conditions; it was even possible to detect the same degradation products in vivo and in vitro for the pigment C.I. 12315 [10]. Two mono-azo pigments (C.I. 12315 and C.I. 12460) showed the same degradation patterns. Bonds were cleaved at the junctions of the azo bridges, and interestingly, a reduction of the azo bridge yielding free amines also occured. Experiments with laser irradiation of similar mono-azo pigments suspended in water, however, only partly revealed analogous degradation products [13]. The presence of a relatively high amount of impurities certainly complicated any verification of photolysis. The choice of solvent or energy dose, however, could be another reason for these discrepancies. In the case of the 3,3′-DCB-based pigments C.I. 21095, C.I. 21108 and C.I. 21110, laser treatment led to the expected reduction of the azo bridge, resulting in the formation of 3,3′-DCB, but not of 3,3′-dichlorodiphenyl.

Regardless of any shortcomings of the described in vitro assays, some reaction pathways are very likely to happen in vivo and sometimes result in the same degradation products, regardless of the irradiation source. Whereas sunlight irradiation leads to continuous, low-level production of degradation compounds, comparable to degradation by enzymes, laser irradiation releases a momentary, high-level spike comparable to concentration spikes of pigment impurities when a tattoo ink is injected into the body. For the carcinogenic aromatic amines identified, toxicological data are available; therefore, worst-case toxicological evaluations using published pigment concentrations in the skin should be possible.

All organic pigments undergo photodegradation. In vitro experiments show that even the visible range of the sun spectrum degrades them to potentially hazardous compounds. It is reasonable to relate this process to in vivo conditions where this range of light penetrates into the dermis. Laser irradiation may also degrade pigments. For some pigments, toxic or carcinogenic degradation substances have been identified. In vitro analytical screenings, even with their inherent shortcomings, have provided evidence that certain azo pigments release carcinogenic aromatic amines like o-toluidine, 2-amino-4-nitrotoluene and 3,3'-dichlorobenzidine under sunlight or laser irradiation. In light of these findings, it is time to consider banning tattoo inks with azo pigments containing carcinogenic aromatic amines or strong allergens in their structure.