The sizes of the pigments in tattoo inks fall into three classes: the black pigments were clearly the smallest, the white pigments the largest and the coloured pigments in between the two. Overall, the results were consistent, with little variation in size within each of the colours from the different suppliers. An exception to this, however, was the yellow inks which contained pigments with a larger range of size distribution. All the blue colours contained the same pigment (P.B.15). Two different green pigments, five different red pigments and six different yellow pigments were used.
Size measurements were performed in water for two reasons: the suppliers add water, and the tattoo pigments are meant to be installed in a watery environment. Some of the tattoo inks provided problems with aggregates when added to water. Of these, some could be disaggregated by sonication and others could not. Differences in the physical dispersion behaviour were observed for the same pigment from different manufacturers (e.g. P.Y.74 from manufacturers 2 and 8), which may indicate differences in the coating of the pigments. Producers may also have added polymers, surfactants and other additives. Common ingredients in the ready-to-use tattoo inks are water, glycerine, alcohol and sometimes preservatives.
The interesting and explicit result is the awareness of the wide use of NPs in tattoo ink. The vast majority of the tested inks contained significant amounts of NPs, except for the white pigments. The black pigments were almost pure NPs. Within these, up to 99·94% of the volume of the material was made up of particles with diameters < 100 nm, with very small mean diameters in one brand, i.e. 41 nm. In general, carbon blacks are available in a wide spectrum of particle sizes, ranging from 5 to 500 nm. The primary aggregates are the characteristic units of carbon black. These are not broken down under normal dispersion conditions. The degree of 'blackness' of the black colours increases with decreasing primary aggregate size.
Carbon black is commonly used in many applications and has been a field of interest in nanotoxicology studies. Carbon black is a component of rubber, plastics, inks and paints, with a global production exceeding 10 million tonnes in 2005. Approximately 90% of carbon black is used in rubber, mainly tyres. The highest exposures to carbon black occur during its production. Previous epidemiological studies have not revealed a clear link between occupational exposure to carbon black and cancer frequency in humans. A study on employees at carbon black plants revealed an increased number of deaths by lung cancer. However, the design was not adjusted for exposure to cigarette smoke. Exposure in industries that use carbon black is difficult to assess because data are sparse. Nevertheless, carbon black is classified by the International Agency for Research on Cancer as possibly carcinogenic to human beings, which is partly based on inhalation studies on rats. Studies exploring the effects of carbon black, involving in vitro and in vivo investigations on animals with particles in the same size range as in the present panel of black tattoo inks, have demonstrated that it induces inflammation and weakly increases the mutant frequency following long-term exposure at a subcytotoxic concentration.[29,31] Damage from carbon black is supposedly a result of formation of reactive oxygen species (ROS). Studies confirm that for materials such as TiO2 and carbon black ultrafine particles (all dimensions < 100 nm) are more toxic and inflammogenic than fine particles. Studies such as these show that the NPs generate ROS to a greater extent than larger particles. It is, however, still important to emphasize that there are presently no studies supporting extrapolations from the effect levels documented in laboratory tests to environmental scenarios, or to tattoos.
With regard to TiO2, the pigment in the white inks, the generated data reveal that the smallest primary particle size is approximately 100 nm. Genotoxicity studies have been carried out to determine the carcinogenic potential of TiO2 NPs with conflicting results. Trouiller et al., among others, suggest that TiO2 NPs are genotoxic in vivo. They studied TiO2 with a primary particle size of 21 nm. Nevertheless, we have not found primary TiO2 particles in such a low size range.
A review on carcinogenicity of azo colorants pinpoints that several epidemiological studies have demonstrated that occupational exposure to benzidine-based dyes used as textile dye has caused bladder cancer in humans. The potential effects of a variation in size of the azo pigments and phthalocyanines used in tattoo inks on human health have not been studied.
Two main issues emerge from the current results. Firstly, is there any unrecognized distribution of tattoo pigments beyond the skin and local lymph nodes? Secondly, should we now re-evaluate known and possible unknown biological effects of tattoos with the now better appreciation of their NPs?
No exposure studies have addressed tattooing as an administrative route of NPs despite the fact that some amount of pigment is implanted intradermally. Approximately 250 mg of azo pigment is required to tattoo a skin area of 100 cm2 (2·5 mg cm−2).
A few reports of tattoo pigment in local draining lymph nodes have appeared in the medical literature, but tattoo pigments have not been reported in any distant organs.[38,39] Engel et al. made a quantitative estimate of the amount of tattoo pigment persisting in the skin of mice. They found that 32% of the pigment had disappeared 42 days after the injection. Biopsies have shown that pigment-laden cells cluster around small vessels presumably in an attempt to eliminate tattoo pigment as in a normal foreign-body reaction through the lymphatic system, bloodstream or the transepidermal route.
Tang et al. investigated the distribution and accumulation of silver NPs in rats with subcutaneous injection. The rats were injected with either silver NPs or silver microparticles (MPs). The silver NPs had a particle diameter of 50–100 nm while the silver MPs had a particle diameter of 2000–20 000 nm. Results indicated that the silver NPs reached the blood circulation and were distributed to the main organs, especially to the kidney, liver, spleen, brain and lung. In contrast, the silver MPs could not invade the blood stream or organ tissue. The authors assumed that physical size determined the distributions. It is reported that the liver and spleen can be considered as the organs that accumulate nanomaterials. A useful field of research might be to perform animal experiments with injection of tattoo ink intradermally with subsequent examination of organs such as the liver and spleen, and the lymph nodes.
The European Commission recently published an extensive report on NPs and the associated risks to humans. Extensive further research is required in order to generate sufficient knowledge for a risk assessment to be compiled and carried out. There is consensus that thorough and accurate particle characterization is an essential part of assessing the potential toxicity of NPs in biological systems. In addition to size there are a number of factors that are implicated in the behaviour and potential effects of NPs. These include shape, composition, state of dispersion, surface area, aggregation state and surface chemistry.[17,27,33] These parameters are currently unknown in tattoo ink.
The concerns raised by some in vitro and in vivo animal studies and weak epidemiological studies are not supported in patients. It is evident that more research is needed before a conclusion can be drawn from the findings in the present study regarding NPs in tattoo inks and risks posed to humans. It is fair to ask why the safety profile of tattoos is so good despite these sizes, which have raised eyebrows in other settings.
Conflicts of interest
We thank Søren Poulsen (R&D project manager) Dyrup A/S, Gladsaxe, Denmark for his help in this project.
The British Journal of Dermatology. 2011;165(6):1210-1218. © 2011 Blackwell Publishing