Ingredients, Chemical Analysis and Safety of Marketed Tattoo Ink Stock Products: Absorption, Distribution, Metabolism and Excretion of Tattoo Colorants and Ingredients in Mouse and Man: The Known and the Unknown

Tattooing is an ancient technique used to stain the skin of humans. Tattoos have been identified in human beings dating back to the Stone Age [1]. In some cultures, like Polynesian tribes, tattooing was an important tool in religion and hierarchy. However, the meaning of tattoos in the western world became ambiguous during the past centuries and was consistently associated with low social status.

Nowadays, tattooing has become very popular worldwide. The current tattoos are black or multi-colored and can be found on almost all parts of the human body. Many tattooed role models, like football, pop or movie stars, have led to a broader cultural acceptance of tattoos. A special category of tattoos is the so-called permanent make-up, in which colorants are placed in the skin to mimic normal make-up [2].

Tattooists usually inject colorants as a suspension into the skin using solid needles, which are actuated by tattoo machines. Tattooists purchase black colorants from tattoo suppliers or through the internet. A fraction of the injected colorant stays in the dermis as particles, and their light absorption within a specific spectral range results in the color of the tattoo. Another fraction of the injected colorant is removed from the skin via the lymphatic or blood vessel systems. As a result, tattoo colorants can be found in the lymph nodes located next to the tattoo [3, 4, 5, 6]. An internet-based survey showed that about 60% of tattoos are either completely or partly black [7].

From a chemical point of view, colorants are classified as either pigments or dyes; however, the chemical structures of pigment and dye molecules are frequently the same. In contrast to dyes, pigments are practically insoluble in the medium in which they are incorporated, and this insolubility is achieved by avoiding solubilizing groups in the molecules, yielding small particles, the so-called pigments. Pigments may be inorganic or organic, colored, white or black materials.

Thus, making a persistent tattoo in skin requires the use of water-insoluble colorants in the form of pigments. In the past, tattooists used inorganic pigments that contained heavy metals such as mercury, chromium or cadmium, resulting in the typical colors yellow (cadmium sulfide), mercury sulfide (red), or chromium oxide (green). Two important inorganic pigments are still in use: Carbon Black for black tattoos and titanium dioxide to reduce the color strength of colored pigments.

Nowadays, colored tattoo colorants mainly consist of organic pigments like azo or polycyclic pigments, which are usually obtained from the chemical industry [8]. These pigments comprise two features perfect for use in tattoos: the pigments exhibit brilliant colors, and they are insoluble in aqueous tissue. The chemistry of black inks, which predominantly contain Carbon Black, has not changed over time. Carbon Black is a powder that mainly comprises amorphous particles of carbon with diameters of a few tenths of nanometers (fig. 1).

Although they are injected into the human body, tattoo colorants usually have no pharmaceutical requirements. Beside the coloring compound (black, red, green, blue, etc.), the colorants may contain other various substances depending on the production methods. The colorants comprise educts, products, and by-products of the respective coloring compound [9]. Different solvents (water, isopropanol, etc.) are used to dissolve the pigment powder, other substances are applied as preservatives, and titanium dioxide is frequently added to change the color strength [8, 10]. Tattoo colorants might also contain various impurities that accidentally got into colorants for unknown reasons. Thus, tattoo colorants usually exhibit a complex mixture of various chemical compounds [11, 12, 13]. At present, the list of identified admixtures and impurities is rather incomplete, and further chemical analysis of the tattoo colorants that are present in the market is required. It is known that the chemistries for colored and black tattoo colorants are rather different, and hence, the colorants may contain different by-products and impurities [8, 11].

Colored pigments are classified by their chemical constitution and can be roughly assigned to azo or polycyclic pigments. Pigments are identified either by their chemical index number or by the pigment shortcut. The azo pigments are subdivided in mono-azo (greenish to medium yellow, reddish yellow to orange), dis-azo (greenish, reddish to orange red), β-naphtol (orange to medium red), naphtol AS (medium red to violet) and metal complex pigments containing nickel, copper or cobalt. The polycyclic pigments are generally condensed aromatic or heterocyclic ring systems. Two important examples are the phthalocyanines (green, blue) and the quinacridone pigments (bluish red, red, violet) [8]. The production of such pigments requires complex chemical synthesis, and the resulting colorant may contain different educts, products and by-products as well as titanium dioxide, to lighten the colorant, and different concentrations of other non-specified compounds.

Colored tattoo pigments like PR.22 can be decomposed by solar radiation [14, 15] or by laser light [16,] yielding decomposition products such as 2-methyl-5-nitroaniline (2,5-MNA), 4-nitrotoluene (4-NT), 2,5-dichloroaniline (2,5-DCA) and 1,4-dichlorobenzene (1,4-DCB) (fig. 1). 4-NT was genotoxic in a human lymphocyte assay [17], and 2,5-MNA, also referred to as 5-nitro-o-toluidine, was shown to cause liver dysfunction in workers from a hair dye factory [18]. Additionally, Sayama et al. [19] showed that 2,5-MNA and some dinitrotoluenes are mutagenic to Salmonella typhimurium YG. Furthermore, 1,4-DCB induced kidney tumors in male rats and liver tumors in male and female mice [20], whereas 2,5-DCA was nephrotoxic in rats [21].

Black colorants are usually produced by the imperfect combustion of hydrocarbons, yielding soot with polycyclic aromatic hydrocarbons (PAHs). The major constituent of black tattoo colorants is Carbon Black, which has been listed by the International Agency of Research in Cancer (IARC) as possibly carcinogenic to humans (group 2B) [22]. High amounts of hazardous PAHs, up to 201 µg/g, were found when analyzing different commercially available black tattoo colorants [11]. PAH molecules are either unbound in the colorant suspension or are attached to the surface of Carbon Black particles. PAHs such as benzo[a]pyrene belong to a large class of well-studied chemical pollutants that ubiquitously occur in the environment. They consist of two or more fused benzene rings and are generated naturally or are found as a result of incomplete combustion of organic materials, fossil fuels, vehicular emissions or even tobacco smoke. Some PAHs are classified by the IARC as human carcinogens (e.g., benzo[a]pyrene), and several others are classified as probably or possibly carcinogenic to humans [23]. The active metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide probably represents the ultimate carcinogen [24].

It is well known that human exposure to complex mixtures of PAHs occurs primarily through three routes: the respiratory tract through the smoking of tobacco products and the inhalation of polluted air, the gastrointestinal tract through the ingestion of contaminated drinking water and food, and skin contact, which usually occurs from occupational exposure [25].

In contrast to topical coal tar application and possible penetration into skin, black colorants are injected into skin during the making of a black tattoo, resulting in an almost complete penetration of PAHs into the skin, after which the substances can distribute throughout the entire body. In addition to their carcinogenic properties, PAHs exert a wide range of deleterious effects towards tissue and cells, including the mutagenesis of oncogenes in skin [26]. PAHs are thus potent immunotoxic agents that impair the functional activation of lymphocytes [27] and inhibit macrophage differentiation [28]. Other analytical investigations revealed the presence of additional hazard substances, such as the softener di-buthyl-phthalate [29]. Another problem may arise from the fact that the particles have small diameters of tenths of nanometers [30]. Thus, the potential of the particles for nanotoxicology should be investigated [31].

Consequently, the skin might react to either colored or black ink suspensions in different manners. Many side effects of tattoos, such as granulomatous, lichenoid or hypersensitive allergic reactions, infections, and malignant skin tumors, have been described in the medical literature [2, 12, 13, 32]; however, the latter might be coincidental [33].

In the western world, most tattooists inject tattoo colorants into skin using the rapidly vibrating needles of a tattoo machine to transport a certain amount of colorant suspension into the skin. The needles thereby leave punctures in the skin that pierce the thin epidermis and may reach the middle of the dermis (fig. 2, left). Experiments with pig and human skin revealed the concentration of, e.g., red pigment, that is placed in skin by such tattoo machines. As tattooing is an archaic procedure, the experiments yielded a concentration range of about 0.60-9.42 mg of pigment per cm² of tattooed skin [34]. The concentrations depended on the size of the pigment crystals, the concentration of pigment applied to the skin surface, the desired color strength of the tattoo, and the tattooing procedure using needles of different sizes and shapes. The mean value was estimated to be about 2.5 mg/cm².

Tattooing damages the skin, causing pain and superficial bleeding. In an internet-based survey, tattooed people reported crust formation, itching, swelling and even superficial infections during the healing process [7]. Being a superficial injury to the skin, the tattooed area should heal within a couple of days. However, the survey also revealed that 8% of the participants still had health problems 4 weeks after tattooing, and 6% had persistent skin problems in the tattooed area. Additionally, 3% stated other health problems such as psychiatric problems and light sensitivity of the tattooed skin. These problems were significantly related to the color of the tattoo, which obviously meant that they were related to the specific chemistry of the colorant. This finding was confirmed by comparing the data of that survey with medical case reports on the location and color of tattoos. The results clearly showed that colored tattoo pigments, in particular red pigments, are mainly responsible for adverse skin reactions and that these reactions occur more often on the extremities [12, 13].

After tattooing skin, pigment particles are exclusively found in the cytoplasm of cells in membrane-bound structures identified as secondary lysosomes [35]. Also, macrophages may contain pigment particles. At first view, the injected tattoo colorants seem to stay in skin forever. However, three major mechanisms may reduce the concentration of colorants that is initially placed in the skin. First, part of the colorant may leave the skin with the bleeding during or directly after tattooing. Second, part of the colorant may be transported away from the skin via the lymphatic or blood vessel system. Third, part of the colorant is decomposed months or years after tattooing because the pigments in the dermis are repeatedly exposed to different light sources, in particular, solar radiation, including UV radiation. Azo pigments are chemically unstable when exposed to UV radiation [14, 15].

Recent investigations provided evidence that the major part of tattoo colorants, such as red pigments (87-99%), disappears from skin months or years after tattooing [36], which could cause a fading of the colored skin. However, when asking tattooed people, almost nobody perceives a change in the tattoo color [7]. The change in the color concentration can be overlooked because of the very high color strength of azo pigments. The decrease in the pigment concentration appears to be very high; however, an in vivo animal model showed that about 30% of intradermally injected PR22 disappeared from skin within 6 weeks after tattooing [37], and up to 60% of tattooed PR22 disappeared when applying excess solar radiation to the animal skin [37]. Thus, the disappearance of such a high fraction of the tattooed pigments is a result of either light-induced decomposition in tattooed skin or pigment transport to other anatomical locations in the body via the lymphatic system.

For example, black tattoo colorants frequently contain substantial amounts of PAHs [11], and enzymatic and non-enzymatic pathways can convert PAHs into hazardous diol-epoxides, such as benz[a]pyrene-7,8-diol-9,10-epoxide [38]. Both pathways might also occur in tattooed skin. First, cytochrome P450-dependent enzymes can be triggered by injecting foreign material, such as tattoo ink, into skin [39]. Second, PAHs in tattooed skin generate singlet oxygen when exposed to UV radiation, leading to oxidation of the PAHs. Akintobi et al. showed the induction of cytochrome P450 1B1 (CYP1B1) expression in human dermal fibroblasts when exposed to xenobiotic substances like 2,3,7,8-tetrachlorodibenzo-p-dioxin [40]. Recent studies have shown that CYP1B1, a newly identified member of the CYP1 family, plays a very important role in the metabolic activation of PAHs [41, 42]. Whether such cytochrome P450-dependent enzymes also play a role in tattooed skin has not been investigated.

Thus, educts, by-products, impurities, and admixtures can be punctured into skin to an unknown extent during tattooing. These compounds may cause various adverse skin reactions, which have been consistently published in the literature [12, 43]. Also, case reports about skin tumors from tattoos have been published and summarized in a recent review article [33]. However, it is still under discussion whether the malignancies, including basal cell carcinoma [44] or malignant melanoma, were coincident.

Tattoo colorants are a rather complex mixture of various compounds that exhibit different chemical and physical structures. The colorants contain pigment particles of different sizes and molecules, and many of the molecules are in the form of monomers, dimers, or polymers with different solubilities, which influences the extent and the route of transport inside the dermis and to other organs. In addition to transport, some constituents of the colorants can be metabolized in skin or in the organs to which the compounds were transported. Thus, tattooing of colorants into skin entails a complex reaction of the skin that triggers the immune system and launches manifold transport processes.

After injection into skin, some of the tattoo pigment particles are encapsulated in the dermis. As mentioned above, the colorants can be transported to other anatomical locations in the body via the blood vessel and lymphatic systems directly after tattooing or via the lymphatic system years or months after tattooing. In particular, a portion of the small particles, admixtures, impurities, as well as educts and products of the pigment molecules may leave the skin directly or during the weeks after tattooing. These transportable compounds have the potential to reach other anatomical locations and might be stored in other organs or may leave the human body via the urinary system or defecation. Thus, organs such as liver, spleen and kidney could be destinations of constituents of tattoo colorants, depending on the route of transport via the lymphatic or blood vessel systems. Consequently, after tattooing the skin, the injected colorants may pose a risk to other organs in the human body. However, except for the transport of tattoo colorants to the lymph nodes (fig. 3) [3, 4, 45, 46, 47, 48], all other transport processes and routes in a human body have been unexplored.

It is assumed that especially large particles, which cannot pass the lymph nodes, stay in the dermis. Thus, any process that reduces the size of the particles will assist in reducing the concentration of pigment particles in skin. A major mechanism for disintegration of the particles is the light-induced decomposition of pigment molecules that may continuously occur whenever tattooed skin is exposed to light sources [14]. Other mechanisms, like enzymatic activities or the recurring activation of macrophages, might contribute to pigment particle transport. However, the contribution of other mechanisms is unknown.

There are currently no indications regarding the systemic effects of tattoo colorants and of their decomposition products because scientific investigations or epidemiologic data are lacking. Millions of people have many and large tattoos [7], with sizes of 600 cm² and more. In cases of such tattoos, about 1,500 mg of, e.g., azo pigments are injected into the human body. In light of the 80% decrease, one should deliberate about whether 1,200 mg of azo pigments and their possible decomposition products could cause health problems in the skin or in the rest of the human body.

In the case of black colorants, substantial amounts of PAHs are injected into skin and hence should be partially transported to other organs. This is of great importance because those PAHs might cause deleterious effects elsewhere in the human body. Recently, placental concentrations of PAHs were analyzed by gas chromatography-mass spectrometry. PAH concentrations above the median, as detected in some cases, were associated with a 4.52-fold increased risk for neural tube defects, a 5.84-fold increased risk for anencephaly, and a 3.71-fold increased risk for spina bifida [49]. PAHs are an important class of environmentally prevalent xenobiotics that can activate oxidative and electrophilic signaling pathways in lymphoid and non-lymphoid cells, including myeloid, epithelial, and other cells [50]. Beside toxicity and mutagenicity [23, 51], PAHs, including the non-classified PAHs such as phenanthrene, are also known to cause immunotoxic effects, particularly IgE regulation [52].

In conclusion, millions of people worldwide have tattoos, for which high amounts of tattoo colorants are injected into the human body. A recent survey revealed that 28% of tattooed individuals have more than four tattoos and that 36% have tattoos that are larger than 900 cm², implying that several grams of tattoo colorants have been injected into skin. Any systemic effects of tattoo colorants, which are administered via tattooing, have not been investigated; thus, we highly recommend performing pharmacological, toxicological and epidemiological studies to clarify the possible impact of tattooing on human health.