Introduction: Pigments of tattoo inks may over time migrate to other parts of the body. Inks kinetics are still poorly understood and little studied. The aim of this first study was to investigate the kinetics of tattoo inks pigment in tattooed porcine skin, which is closer to human skin than mouse skin studied in the past. Methods: Three animals were tattooed on the inner thigh and one animal served as untreated control. Skin biopsies were taken on days 7, 14, and 28 after tattooing. Animals were sacrificed on day 28 and homogenate samples of the liver, spleen, kidney, and brain, as well the local lymph nodes were prepared. All samples were analyzed for ink components using inductively coupled plasma-mass spectrometry. The ink itself was characterized by dynamic light scattering and matrix-assisted laser desorption-ionization mass analysis. Results: Titanium (212 g/kg), copper (6 g/kg), aluminum (1 mg/kg), zirconium (1 mg/kg), and chromium (3 mg/kg) were found in the ink. Significant deposits of ink elements were detected in the tattooed skin when compared to non-tattooed skin from the same animal (mean ± standard deviation: titanium 240 ± 81 mg/kg, copper 95 ± 39 mg/kg, aluminum 115 ± 63 mg/kg, zirconium 23 ± 12 mg/kg, and chromium 1.0 ± 0.2 mg/kg; p < 0.05). Lymph node concentrations of titanium, copper, aluminum, zirconium, and chromium were 42 ± 2 mg/kg, 69 ± 25 mg/kg, 49 ± 18 mg/kg, 0.3 ± 0.2 mg/kg, 0.5 ± 0.2 mg/kg, respectively. Conclusion: Deposits in skin were unchanged from days 7–28 indicating no redistribution or elimination. No significant deposits of ink elements were found in the liver, spleen, kidney, and brain. In conclusion, our findings confirmed distribution of elements from tattoos to regional lymph nodes, but neither to excretory organs, e.g., liver and kidney, nor to spleen and brain. Thus systemic internal organ exposure was not found.

About 20% of the world population is tattooed, thus the exposure to tattoo inks, which can undergo absorption and systemic distribution, is huge [1, 2]. Apart from serving as body art, nowadays, tattoos are also used in medical applications such as nipple areola reconstructions following a mastectomy and breast reconstruction [3]. Given the potential presence of hazardous compounds in the ink, in 2020 the European Union implemented a new regulation on the chemical composition of tattoo inks binding to all member states; however, regulation of tattoo ink products in other countries is still scattered [4‒6].

There is a range of tattoo complications occurring directly in tattoos, but sometimes also with systemic manifestations [7]. Indeed, allergy in red tattoos related to organic azo pigment is a special challenge, however, also metal wear from needles might also cause allergy [8].

Beside this, the state of tattoo ink biokinetics, which was recently reviewed is basically still showing a lack of knowledge [9]. Indeed the metabolism of pigment has been only studied in rat and human microsomal proteins [10]. Bäumler [11] demonstrated that up to 60% of tattoo pigments disappear from mice skin within 6 weeks after tattooing and solar irradiation, which indicates a systemic distribution. Another study in mice found tattoo ink particles in the Kupffer cells of the liver after 1 year [12]. Therefore, the questions and concerns about tattoo ink biokinetics have been discussed over the years, especially since tattoo inks have been shown to contain potentially carcinogenic polycyclic aromatic hydrocarbons (PAHs) which were found in lymph nodes of tattooed mice [4, 13‒15]. Furthermore, tattoo-colored lymph nodes have been found by chance in human, for example, during autopsies [16].

However, rodent skin is thin and hard to tattoo offering an unrealistic substitute or model of human skin. Hence, in the present study, we used porcine skin, which has been demonstrated to closely resemble human skin. Since porcine skin shows major anatomical site variation, we concentrated on the skin of the inner thigh of the pig, which shows the closed similarity to human skin in thickness as well as texture. In this prospective study, we investigated the biokinetics of tattoo ink pigments over 28 days post-tattooing to measure ink migration from the skin to the lymph nodes and internal organs.

Animals

Animal study protocols were approved by the Austrian Federal Ministry of Education, Science and Research (BMBWF, application no. 2020-0.193.102) in accordance with the 2010/63/EU directive on the protection of animals used for scientific purposes. Four female pigs (age at study entry: 8 weeks, average weight with standard deviation 19.6 ± 2.7 kg) were used in this study. Prior to any study-related activity, the animals were acclimatized at the Institute for Biomedical Research of the Medical University of Graz for 10 days. They were housed in groups of 2 and had access to food and water for the entire duration of the experiment. All animal handling was carried out by veterinarians and/or other qualified personnel.

Tattoo Inks

After analysis of 10 different colors with inductively coupled plasma-mass spectrometry (ICP-MS), we selected the ink color with the highest titanium content (Periwinkle, Eternal Ink EUROPE GmbH, Germany) for use in this prospective study. Furthermore, this ink was analyzed by dynamic light scattering analysis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry.

Schedule

The trial began in April 2022. Over the course of 4 weeks, the animals were anesthetized 4 times. On day 1, shaving and tattooing were performed. Punch biopsies were taken on days 7, 14 and 28. After sacrificing the animals on day 28, regional lymphadenectomy was performed and the internal organs of interest were removed. The detailed procedure of anesthesia, euthanasia, tattooing, and organ removal can be found in the online supplementary material S1 (for all online suppl. material, see https://doi.org/10.1159/000536126).

Tissue Samples

All skin and organ biopsies were taken with 6 mm biopsy punch (Kai Medical, Honolulu, HI, USA). Skin biopsies were collected under general anesthesia, biopsy sites were sutured with a single stitch Dafilon 2/0 (B|Braun, Melsungen, Germany), and surgical wounds covered with medical dressing as described in the online supplementary material S2.

On day 28, extracorporeal circulation was instituted and the liver, spleen, kidneys, lymph nodes, and brain were carefully removed without damaging the primary vessels. Once all organs were removed, they were further cannulated in the main artery and perfused with additional 0.9% NaCl solution (4°C) in order to wash out as much residual blood as possible.

Punch biopsies were then taken from each liver, spleen, and kidney segment. Biopsies were also taken from each side of the brain, respectively, and from the frontal, temporal, and occipital lobes. After weighing the organs and samples, the remaining tissue was homogenized and samples were stored at −80°C. Samples were analyzed for ink components by ICP-MS. The detailed homogenization process is described in the online supplementary material S2.

Analytical Methods

Analytical methods applied, e.g., dynamic light scattering, ICP-MS, and MALDI mass spectrometry are described in online supplementary material S1.

Statistical Analysis

For descriptive statistics, all data are presented as mean ± standard deviation, unless otherwise stated. Element concentration in skin samples was analyzed by the Mann-Whitney U test for nonparametric variables. The test was performed on tattooed skin versus non-tattooed control skin at days 7, 14, and 28. Since multiple samples of tattooed as well as non-tattooed animals of lymph nodes were available Mann-Whitney U test for nonparametric variables were performed. p values below 0.05 were considered statistically significant. Since only one sample per animal was available for the homogenized organs, we were not able to perform any statistical analysis on the homogenates. All analyses were performed with GraphPad Prism 9 software (San Diego, CA, USA).

The product Periwinkle ink was selected due to its high titanium concentration, facilitating tracking of this element in the tissues. The chemical composition and pigment particle size in the ink are shown in Tables 1 and 2.

Table 1.

Dynamic light scattering (DLS) size distribution of particle diameter in the ink batches

Z-Average (d.nm)SDPdlMean Peak 1, Mean Int (d.nm)Mean Peak 1, Mean Int SD
Periwinkle 339.4 8.4 0.2 405.4 19.9 
Periwinkle (TiO2 fraction) 389.3 16.8 0.2 427.0 28.3 
Periwinkle (Blue fraction) 61.3 2.3 0.3 78.8 3.5 
Z-Average (d.nm)SDPdlMean Peak 1, Mean Int (d.nm)Mean Peak 1, Mean Int SD
Periwinkle 339.4 8.4 0.2 405.4 19.9 
Periwinkle (TiO2 fraction) 389.3 16.8 0.2 427.0 28.3 
Periwinkle (Blue fraction) 61.3 2.3 0.3 78.8 3.5 

Data are displayed as mean from three consecutive measurements with automatic run and duration mode.

SD, uncorrected sample standard deviation; PdI, polydispersity index; Int, intensity.

Table 2.

Periwinkle tattoo ink composition

Periwinkle
Ti 211.49±3.79 g/kg 
Al 1.19±0.11 g/kg 
Cu 5.68±0.01 g/kg 
Fe 3.14±0.06 g/kg 
Ni 3.30±1.70 mg/kg 
Zr 1.29±0.02 g/kg 
Mo 23.00±0.00 mg/kg 
Sn 10.0±0.20 mg/kg 
Nb 8.60±0.20 mg/kg 
Cr 3.00±0.03 mg/kg 
2.08±0.03 mg/kg 
Hf 28.30±0.10 mg/kg 
Periwinkle
Ti 211.49±3.79 g/kg 
Al 1.19±0.11 g/kg 
Cu 5.68±0.01 g/kg 
Fe 3.14±0.06 g/kg 
Ni 3.30±1.70 mg/kg 
Zr 1.29±0.02 g/kg 
Mo 23.00±0.00 mg/kg 
Sn 10.0±0.20 mg/kg 
Nb 8.60±0.20 mg/kg 
Cr 3.00±0.03 mg/kg 
2.08±0.03 mg/kg 
Hf 28.30±0.10 mg/kg 

Ti, titanium; Al, aluminum; Cu, cooper; Fe, iron; Ni, nickel; Zr, zirconium; Mo, molybdenum; Sn, tin; Nb, niobium; Cr, chromium; V, vanadium; Hf, hafnium.

The label declares the use of the white pigment titanium dioxide, pigment red 22, and Carbon Black; however, the ink appeared blue. Although not labeled on the bottle, the analysis by MALDI showed copper phthalocyanine blue (CI 74160) [17]. Furthermore, MALDI analyses did not detect the labeled pigment red 22 (Fig. 1). To illustrate auxiliary materials used for wound care during the tattooing process as a source of elementary permeation while the skin in a state of healing with broken barrier, we tested all relevant care products applied during the study. The results are shown in Table S1 in online supplementary material S3, demonstrating that care products were not a source of titanium or copper permeation.

Fig. 1.

MALDI analysis for detection of organic pigments. a Periwinkle ink used for tattooing in the study with zoom to the m/z (mass to charge ratio) between 565 and 585. b Reference spectrum of copper phthalocyanine blue (CI 74160) with zoom to the m/z (mass to charge ratio) between 565 and 585. The same specific mass pattern can be seen in the ink in (a). c Reference spectrum of pigment red 22 (CI 12315) declared on the ink label. None of the characteristic main peaks appear in the spectrum in (a).

Fig. 1.

MALDI analysis for detection of organic pigments. a Periwinkle ink used for tattooing in the study with zoom to the m/z (mass to charge ratio) between 565 and 585. b Reference spectrum of copper phthalocyanine blue (CI 74160) with zoom to the m/z (mass to charge ratio) between 565 and 585. The same specific mass pattern can be seen in the ink in (a). c Reference spectrum of pigment red 22 (CI 12315) declared on the ink label. None of the characteristic main peaks appear in the spectrum in (a).

Close modal

Punch biopsies from tattooed skin showed significant amounts of elements delivered with the ink of the elements directly in the tattooed skin at any time (7, 14, and 28 days). With chromium at days 7 and 28 as exception, all concentrations were statistically significantly (p < 0.05) increased versus non-tattooed skin, as shown in Figure 2. The highest absolute difference between the average element concentrations in the tattoos versus non-tattooed control was found for titanium with a total difference of +238.0 mg/kg in punch biopsies from tattoos, followed by aluminum (+108.4 mg/kg), copper (+92.3 mg/kg), iron (+59.9 mg/kg), zirconium (+22.6 mg/kg), hafnium (+0.5 mg/kg), chromium (+0.4 mg/kg), and molybdenum (+0.1 mg/kg). Measured levels were remarkably constant remained constant with no significant decline from day 7 to day 28.

Fig. 2.

Element analysis with ICP-MS of skin biopsies at day 7, 14, and 28. Statistical analysis were performed between tattooed group (n = 6) versus control (n = 3) using the Mann-Whitney U test for nonparametric variables (*p value <0.05).

Fig. 2.

Element analysis with ICP-MS of skin biopsies at day 7, 14, and 28. Statistical analysis were performed between tattooed group (n = 6) versus control (n = 3) using the Mann-Whitney U test for nonparametric variables (*p value <0.05).

Close modal

Pigment deposition in the lymph nodes was macroscopically visible at end of study day 28 (Fig. 3). Analysis of lymph node samples showed the same elements as in the tattoo ink product. Except for hafnium and molybdenum, all concentrations were statistically significantly increased (p < 0.05) compared with lymph nodes of the control animal (Fig. 4). Elements measured in the organ samples of the brain, liver, spleen, and kidney, were not notably different in comparison to the control organ samples, as represented in Figure 4 and Table S2 in online supplementary material S3. The largest absolute increase in the average element concentrations in lymph node samples versus control was observed for iron with a total difference of +218.9 mg/kg, followed by copper (+68.2 mg/kg), aluminum (+48.4 mg/kg), titanium (+41.6 mg/kg), chromium (+0.3 mg/kg), zirconium (+0.2 mg/kg), molybdenum (+0.1 mg/kg), and hafnium (+0.0 mg/kg).

Fig. 3.

Regional lymph node of the groin after sacrifice at day 28.

Fig. 3.

Regional lymph node of the groin after sacrifice at day 28.

Close modal
Fig. 4.

Element analysis with ICP-MS of peripheral organs (lymph nodes, liver, kidney, spleen, and brain) at day 28. Statistical analyses of lymph node samples were performed between tattooed group (n = 9) versus control (n = 3) using the Mann-Whitney U test for nonparametric variables (*p value <0.05).

Fig. 4.

Element analysis with ICP-MS of peripheral organs (lymph nodes, liver, kidney, spleen, and brain) at day 28. Statistical analyses of lymph node samples were performed between tattooed group (n = 9) versus control (n = 3) using the Mann-Whitney U test for nonparametric variables (*p value <0.05).

Close modal

The data from our study demonstrate significant deposition of various ink elements in the regional lymph nodes as well as in the skin, but not in any other organ. Since no previous study has examined the biokinetics of ink in vivo in a porcine model this represents the first of its kind.

The study period of 28 days represents the entire normal healing phase until long-term steady-state biokinetics is established. Although we could not detect pigment-derived particles in peripheral organs, distribution of smaller particles may theoretically also occur over a prolonged time, well beyond 28 days. The steady-state biokinetics observed that elementary deposits were insoluble. However, limit of detection of elements in internal organs does not exclude that minute amounts might have reached these organs. Indeed, Sepehri et al. [12] detected pigments particles in mice liver by electron microscopy.

A tattoo of approximately 9% of the total pigs’ skin surface was chosen as a realistic provocatory scenario representing a larger tattoo made in one session. However, the tattoo/skin surface ratio became smaller with the growth of the animals, whose weight almost doubled during the study period.

Interestingly, ink component concentrations in the skin remained stable between days 7, 14, and 28. The study indicates that local and regional distribution of elements from the ink takes a few days only, followed by steady state biokinetics already after this early point. We believe that this steady state continues for months or years beyond day 28. However, this assumption cannot be extrapolated to other chemical substances or organic pigment particles, depending on solubility or chemical decomposition by sunlight or metabolism.

Copper concentrations in the lymph nodes of tattooed animals were up to 60 times higher when compared to non-tattooed animals. Copper limits published in the European tattoo ink restriction only apply to soluble copper which does not have such a prolonged retention in skin and lymph nodes after tattooing [5]. The copper found in this analysis is most likely bound to the copper phthalocyanine pigment shown to be present in the ink.

In accordance with the literature, our study showed that a major part of tattoo ink elements migrate from the injected skin site to the local lymph nodes – including titanium dioxide with a mean size of >300 nm. Others have shown that even larger particles were transported to the lymph nodes [8]. We observed an increased ratio of copper/titanium concentration in the lymph nodes compared to the skin (1.6 vs. 0.4) which indicates smaller copper phthalocyanine particles are more prone to migrate than the larger titanium dioxide particles. However, the ratio of titanium and copper is already smaller in the skin compared to the original ink (about 38). Since we did not monitor particle excretion via wound healing, we cannot answer how this change of Ti/Cu ratio occurred.

We did not find a significant deposit of ink elements in other organs besides the lymph nodes which as mentioned are contrary to the findings of Sepheri et al. [12] who found ink pigments in the liver of tattooed mice. This discrepancy may be related to the method used or species difference. Mice skin is much thinner than porcine skin and therefore more easily penetrated by tattoo needles resulting in deep deposition of pigments. However, pig skin is structurally much closer to human skin, and therefore a better model when tattoo pigment shall be installed by the tattoo needle and machine in a way directly comparable to tattooing as performed in humans. Furthermore, the pig is the preferred model for human wound healing studies [18, 19]. Interestingly, our results show an apparent tendency of accumulation of zirconium in the kidney of tattooed animals when compared to non-tattooed. However, this tendency was not confirmed since we were not able to perform any statistical analysis.

Biokinetics of tattoo pigments under natural conditions may be influenced by external factors such as the sun, which may cause photochemical breakdown of pigments. Tattoo removal by lasers may lead to higher deposits in the local lymph nodes and at least in theory to increased distribution to internal organs [20]. To achieve a more detailed understanding of tattoo ink pigment biokinetics and possible long-term effect through ink organ deposition, future studies in our pig tattooing model with larger cohort sizes and longer observational time need to be performed.

A significant deposit of tattoo ink elements was detected in the skin and lymph nodes but no other peripheral organs investigated.

We thank Prof. Dr. Philipp Stiegler and the entire Division of Transplantation Surgery of the Medical University of Graz for their contributions during the organ explanation. Lastly, we would like to thank Sandra Losinschek, Sarah Victoria Wünscher, Adrian Mogl, and Michael Dirks for their precious help during the entire trial.

This study protocol was reviewed and approved by the Austrian Federal Ministry of Education, Science and Research, approval number BMBWF 2020-0.193.102.

There is no financial interest in any of the products, devices, or drugs discussed in the manuscript. All the authors declare no conflict of interest.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

J.C.-D., H.L., S.M., J.F., I.S., T.R., W.G., P.K., and L.-P.K. contributed to the study conception and design and have been involved in the writing of the manuscript and have helped in drafting the article or revising it critically for important intellectual content. They have also seen and approved the final version of the manuscript and state that if the article is accepted, it will not be published elsewhere.

The data that support the findings of this study are not publicly available due to ethical animal study guidelines but are available from the corresponding author Dr. Cambiaso Daniel upon reasonable request.

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