Highly stable and safe biocompatible black tattoo ink based on Pluronic polymer-coated carbon black nanoparticles

Black is the most common tattoo color, but only a few studies have shed light on the multitude of functional and contaminating chemicals present in black inks. These studies have generally shown that black inks are a diverse group, containing anything from 5 to 50+ organic components. Little is known about the possible effects on humans of internalizing these chemicals. Analysis has shown that the production of the main component, carbon black, can lead to the formation of pigments with polycyclic aromatic hydrocarbon (PAH) contents that range from very high to almost completely absent. Similar variations in PAH concentrations are observed in black inks. PAHs are known carcinogens and thus, low recommended levels have been suggested by the Council of Europe. Reactive oxygen species (ROS) have recently been a topic in scientific literature related to tattoo ink. Again, it has been shown that some inks produce deleterious ROS (e.g. singlet oxygen or peroxyl radicals), presumably via either adhered organic compounds or particle surface defects. It has been shown that black tattoo inks may contain a multitude of chemicals, including carcinogens and allergens, and some have unknown toxicologies. However, it has additionally been demonstrated that some black inks already on the market do not produce ROS and also contain PAHs at levels that are below those recommended by the Council of Europe and very few additional contaminants.

Introduction: Sonographic evaluation of the axilla and percutaneous biopsy of abnormal lymph nodes with fine needle aspiration (FNA) or core needle biopsy (CNB) has become more common practice in patients with newly diagnosed breast cancer prior to neoadjuvant chemotherapy (NAC). Sentinel lymph node (SLN) biopsy is considered the gold standard for axillary staging in clinically node negative breast cancer patients. Currently, there is no clear correlation of sonographically detected abnormal lymph nodes and open surgical assessment. We conducted an exploratory pilot study which marked suspicious axillary lymph nodes with black tattoo ink at the time of percutaneous needle biopsy prior to NAC. Black nodes visualized during axillary surgery were evaluated in comparison to SLNs. Methods: Breast cancer patients with clinical and/or sonographically suspicious axillary lymph nodes prior to NAC were included in the study. Following FNA or CNB biopsy of node, 0.1 to 0.5 ml of a sterile, highly purified, biocompatible fine carbon suspension (Spot™) was injected into the cortex of the lymph node and adjacent soft tissue. A total of 12 patients were injected with black ink prior to NAC. Intraoperative presence of black pigment was assessed and correlation between sentinel and tattooed nodes were evaluated. Results: Nine patients had a positive percutaneous lymph node biopsy prior to NAC. The average number of days that elapsed between injection and to surgery was 130 days. A successful SLN procedure was performed in all patients. A black tattooed node was identified in all patients and correlated to a SLN. 7 patients were down-staged in the axilla and 6 patients did not go onto completion axillary dissection. One patient with a negative SLN had a completion axillary dissection, but no additional positive lymph nodes were found. Four patients with positive SLN had a completion axillary dissection (1 of whom was a false negative percutaneous biopsy). In all four patients, the positive sentinel node contained visible black ink. There was one patient who had an additional positive sentinel node, which was not black. Two axillary dissections contained additional positive nodes. Conclusion: Black ink tattooing with sterile black ink (Spot™), successfully marked suspicious lymph nodes prior to NAC. These correlated to a SLN. In node positive patients with a partial response in the axillary lymph nodes following neoadjuvant chemotherapy, previously marked, black-inked node proved to be the persistent positive node. Tattooing of lymph nodes at the time of percutaneous biopsy may improve the accuracy of surgical axillary staging by aiding in the intra-operative identification of previously biopsied nodes. Citation Format: Nicole Choy, Jafi Lipson, Sunita Pal, Debra Ikeda, Long Trinh, Kimberly Allison, Michael Ozawa, Amanda Wheeler, Irene Wapnir. Correlation of percutaneously biopsied axillary lymph nodes marked with black tattoo ink prior to neoadjuvant chemotherapy with sentinel lymph nodes in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-01-05.

Black tattoo inks are composed of carbon nanoparticles, additives and water and may contain polycyclic aromatic hydrocarbons (PAHs). We aimed to clarify whether reactive oxygen species (ROS) induced by black inks in vitro is related to pigment chemistry, physico-chemical properties of the ink particles and the content of chemical additives and contaminants including PAHs. The study included nine brands of tattoo inks of six colours each (black, red, yellow, blue, green and white) and two additional black inks of different brands (n = 56). The ROS formation potential was determined by the dichlorofluorescein (DCFH) assay. A semiquantitative method was developed for screening extractable organic compounds in tattoo ink based on gas chromatography-mass spectrometry (GC-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Two black inks produced high amounts of ROS. Peroxyl radicals accounted for up to 72% of the free radicals generated, whereas hydroxyl radicals and H₂O₂ accounted for <14% and 16%, respectively. The same two inks aggregated strongly in water in contrast to the other black inks. They did not exhibit any shared pattern in PAHs and other organic substances. Aggregation was exclusively shared by all ink colours belonging to the same two brands. Ten of 11 black inks had PAH concentrations exceeding the European Council's recommended level, and all 11 exceeded the recommended level for benzo(a)pyrene. It is a new finding that aggregation of tattoo pigment particles correlates with ROS production and brand, independently of chemical composition including PAHs. ROS is hypothesized to be implicated in minor clinical symptoms.

The mechanisms responsible for variable responses of cosmetic tattoos to Q-switched laser removal treatment remain unclear. We sought to investigate the properties of tattoo inks that may affect the efficacy of laser-assisted tattoo removal. The absorption of white, brown, and black inks before and after Q-switched neodymium-doped yttrium aluminum garnet laser irradiation were analyzed by a reflectance measurement system. Rats were tattooed using the three inks and treated with the same laser for two sessions. Skin biopsies were taken from the treated and untreated sites. Black ink showed strong absorption, reduced after laser irradiation, over the entire spectrum. White ink had low absorption over the visible light spectrum, and brown ink had strong absorption at 400-550 nm wavelengths. White and brown inks turned dark after laser exposure, and the absorption of laser-darkened inks were intermediate between their original color and black ink. White, brown, and black tattoos in rat skin achieved poor, fair to good, and excellent responses to laser treatment, respectively. Transmission electron microscopy showed that white tattoo particles were the largest, brown were intermediate, and black were the smallest before laser. After laser treatment, white and brown tattoo particles were mixtures of large and small particles, while black particles showed overall reduction in number and size. Black tattoo ink's excellent response to Q-switched lasers was associated with its strong absorption and small particle size. White tattoo ink's poor response was associated with its poor absorption, even after laser darkening, and large particle size.

BackgroundDuring the last decade, the number of people with ≥1 tattoo has increased noticeably within the European population. Despite this, limited safety information is available for tattoo inks.ObjectivesTo test the skin sensitization potential of 5 tattoo inks in vitro by using reconstructed human skin (RHS) and the contact sensitization biomarker interleukin (IL)‐18.MethodsTwo red and 3 black tattoo inks, 1 additive (Hamamelis virginiana extract) and 1 irritant control (lactic acid) were tested. The culture medium of RHS (reconstructed epidermis on a fibroblast‐populated collagen hydrogel) was supplemented with test substances in a dose‐dependent manner for 24 hours, after which cytotoxicity (histology; thiazolyl blue tetrazolium bromide assay) and skin sensitization potential (IL‐18 secretion; enzyme‐linked immunosorbent assay) were assessed.ResultsAll but 1 ink showed cytotoxicity. Notably, 1 red ink and 1 black ink were able to cause an inflammatory response, indicated by substantial release of IL‐18, suggesting that these inks may be contact sensitizers.ConclusionsThe in vitro RHS model showed that 4 tattoo inks were cytotoxic and 2 were able to cause an inflammatory IL‐18 response, indicating that an individual may develop allergic contact dermatitis when exposed to these tattoo inks, as they contain contact sensitizers.

The incidence of clinical complications such as granuloma formation and sarcoidosis is often seen in black tattoos and may be associated with agglomeration of black pigment. To measure count and dimensions of agglomerates in black tattoo inks vs red inks and to compare old inks and new inks of identical brands. Examination was performed by light microscopy (Olympus BX51™ ) with magnification 40X, immersion oil. Photographs (Jenoptik Gryphax RGB camera) were taken of each ink sample and analysed by ImageJ software; count, area, width, height, circumference and circularity index were measured. Agglomerates were defined as width and height of objects above 800nm. Twenty-one new unopened black inks and 17 new unopened red inks were compared. Furthermore, five old black inks and five old red inks, that had been opened and stocked for over 2years, were compared with new products of the same brands. Black agglomerates were area wise and with respect to width, height and circumference significantly larger compared with red agglomerates and more circularly shaped. Count of agglomerates was lower in black inks than in red inks, in accordance with bigger dimensions of black agglomerates. Comparison of old and new inks indicated old inks have larger agglomerates but variable bottle size and storage conditions may have confounded results. Pigment agglomerates in black tattoo ink stock products were sized larger than agglomerates in red inks. Agglomerates found directly in black inks may predispose to granuloma formation in black tattoos causing sarcoid reaction.

Black tattoo ink comprises hydrophobic carbonblack nanoparticles. We hypothesized that black tattoo inkdemonstrates transient dynamic activity in an ultrasound field. Brightness-mode sonography was performed on cylindrical receptacles of different bore diameters, filled with black tattooink, water, saline, or air, using pulsed ultrasound with center frequencies of 13 MHz and 5 MHz. The scattering from black ink itself lasted less than tenminutes. At 13-MHz sonication, a transient drop in sound speed was observed, as well as a transient lessening of scattering from distal phantom tissue. The linear acoustic attenuation coefficient of pure black ink was measured to be 0.15±0.01 dB cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">−1</sup> MHz <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">−1</sup> , equal to whole blood. Low-intensity ultrasonic tattoo removal would be of interestas an alternative to techniques that damage surrounding tissue.

Because it is difficult to totally remove ink particles trapped in the skin, tattoo removal-especially for deeply and densely pigmented types-remains a substantial issue. This frequently leads to scarring, persistent pigmentation, or an incomplete removal. Significant promise in resolving this problem since high-peak power pulsed Nd: YAG lasers can efficiently target ink particles even in deeper skin layers, they have shown. The purpose of this study is to assess how well the Nd: YAG laser uses tissue photodisruption to remove deeply embedded tattoo ink. High intensity Nd: YAG lasers provide a very efficient way to remove tattoos by dissolving ink particles with the least amount of harm to surrounding tissue. Two sessions of Q-switched Nd: YAG (1064nm) laser treatment was performed on a 36-year-old woman who had a very deep black tattoo on her arm. When the tattooed area was treated with a laser beam with a 6-mm spot size and 5J/cm² fluence. After two months, the process was repeated, and the last evaluation was carried out one month after the second therapy. For histologic research objectives, one local white rabbit was also included in this investigation. Under general anesthesia, the rabbit was injected with tattoos of black pigment and given a single session. Black ink significantly lightened without leaving any scars and a crust formed right after. The spectroscopic properties of black tattoo ink were examined. Following laser exposure, there was a noticeable decrease in the appearance of the ink without any indication of inflammation or cell growth. Within ten days, the skin's texture improved and the tattoo was 85% cleared after two laser sessions without blistering or changes in texture. Black ink granules were efficiently broken down by laser-induced cavitation, creating structures resembling bubbles. Ink fragments were phagocytized and distributed throughout the layers of the skin, whereas histological examination of treated black tattooed rabbit skin revealed a notable decrease in ink particle size without inflammatory reactions. albeit there was no injury to the epidermis, the 1064nm Nd: YAG laser produced deep vacuoles and selective absorption by black ink, albeit localized inflammatory reactions were noted. The 1064nm Q-switched Nd: YAG laser, operating at 6ns, demonstrated safety and effectiveness for deep tattoo removal. It achieved 85% clearance after two sessions, without inducing pigmentation changes.

This study aimed to analyze black tattoo inks by means of energy dispersive spectroscopy and backscattered scanning electron microscopy. The sample consisted of five types of commercial tattoo pigments of the black colour (Easy Glow™, Electric Ink™, Iron Works™, Master Ink™, and Viper™). An Energy Dispersive Spectroscopy (EDS) detector (Silicon Drift Detector - SDD - type) attached to a Scanning Electron Microscope (SEM) device (Tescan Vega3 LMU, Libusina, Czech Republic) was used. X-ray characteristic signs were detected for each tattoo ink in an interval between 0 and 2.5keV. The electron acceleration potential in the microscope was 15keV. Two regions were analyzed for each sample (n = 10). On each region, a micrography of backscattered electrons (BSE) was obtained. Means and standard deviations (SD) of the weight percentages (Wt%) were calculated. C and O were predominant, with a mean O/C ratio between 2.69 and 2.74 Wt%. Electric Ink and Master Ink were the most similar pigments, while Easy Glow was the most distinctive - with agglomerates of Al that had a concentration 25 × higher than other specimens. Other compounds detected in the sample were Cl and Cu. EDS and SEM were efficient to distinguish black tattoo inks. These are our preliminary outcomes on the use of EDS and SEM to analyze black tattoo inks. Thus, careful interpretation is necessary to avoid rash applications in human identification practice.

Bioanalytical evidence that chemicals in tattoo ink can induce adaptive stress responses

A 61-year-old man presented with a 2-week history of a rash affecting tattooed skin. Following an initial 48-h period of fevers, a papular eruption developed within tattoos. Within 1 week it significantly progressed and was associated with itch and skin hyperalgesia. He had a background of BRAF-mutant resected stage III malignant melanoma, for which he was taking dabrafenib and trametinib daily starting 5 months earlier. He had an excellent performance status and no evidence of disseminated metastatic disease on updated imaging. His only other medication was perindopril. He had no history of dermatological issues or any relevant dermatoses in his family history. Review of systems was noncontributory. On examination, there were erythematous papules and plaques confined to the black ink of tattoos, with slight extension beyond the tattoo boundaries and secondary desquamation. Coloured ink was unaffected. There was no mucosal involvement, blistering, skin fragility, ulceration or evidence of infection. Skin biopsy demonstrated granulomatous inflammation surrounding black pigmented material. Laboratory tests including full blood count, inflammatory markers, and renal and liver profiles were normal. Antinuclear antibodies were negative. Serum angiotensin-converting enzyme was low, and no pulmonary findings were noted on imaging. Dabrafenib and trametinib were paused after the initial fever. Clobetasol propionate ointment was initiated to treat affected areas topically. BRAF and MEK inhibitor therapy (BRAF/MEKi) was recommenced at a reduced dose 4 weeks later when the rash had resolved. Subtle dermal thickening within black tattoo ink was noted within 1 week of restarting therapy, although this subsided, and the patient has continued on BRAF/MEKi without symptoms since. Few cases of cutaneous tattoo reactions to BRAF/MEKi have been reported. They have been recognized in those on immunotherapy, as a manifestation of immune restoration syndrome in the context of antiretroviral therapy for HIV, and in sarcoidosis (Kluger N. Tattoo reactions associated with targeted therapies and immune checkpoint inhibitors for advanced cancers: a brief review. Dermatology 2019; 235: 522–4). In previously reported cases of tattoo reactions due to BRAF/MEKi, like in this case, it was particularly black ink affected, and most patients tolerated treatment recommencement. The pathophysiology is not fully understood, although it may represent innate immune ­system activation, T-cell dysfunction, or BRAK/MEKi-induced paradoxical granuloma formation through activation of the AKT/mTOR pathway (Kluger). Distinguishing sarcoidosis-associated tattoo reactions from other granulomatous tattoo reactions can be challenging, although it has been reported that the extension of erythema beyond tattoo borders is less frequently seen in sarcoidosis (Kluger). In summary, this case illustrates a rare manifestation of reaction to targeted therapy for melanoma, which may be more frequently encountered as therapeutic indications expand for neoadjuvant use and in earlier stages of disease.

Tattooing has recently become increasingly popular. Using tiny needles, tattooists place the tattoo ink in the dermis along with numerous unknown ingredients. Most tattoos consist of black inks, which are predominantly composed of soot products (carbon black with polycyclic aromatic hydrocarbons). Black tattoos cause skin problems, including allergic reactions, but the responsible substance frequently remains unknown. We applied gas chromatograph-mass spectrometry analysis to search for hazardous compounds in 14 different commercially available black tattoo ink samples. The analysis revealed that all inks contained the softener substance dibutyl phthalate (0.12-691.2 µg/g). Some of the inks contained hexachloro-1,3-butadiene (0.08-4.52 µg/g), metheneamine (0.08-21.64 µg/g), dibenzofuran (0.02-1.62 µg/g), benzophenone (0.26-556.66 µg/g), and 9-fluorenone (0.04-3.04 µg/g). The sensitizing agent dibutyl phthalate acts directly on keratinocytes and can drive Th2 responses following skin exposure via induction of thymic stromal lymphopoietin gene expression. Hexachloro-1,3-butadiene is genotoxic in vitro and 9-fluorenone is cytotoxic, generating reactive oxygen species under light exposure. The substances found in the inks might be partially responsible for adverse skin reactions to tattoos.

In the past years, tattoos have become very popular worldwide, and millions of people have tattoos with mainly black colours. Black tattoo inks are usually based on soot, are not regulated and may contain hazardous polycyclic aromatic hydrocarbons (PAHs). Part of PAHs possibly stay lifelong in skin, absorb UV radiation and generate singlet oxygen, which may affect skin integrity. Therefore, we analysed 19 commercially available tattoo inks using HPLC and mass spectrometry. The total concentrations of PAHs in the different inks ranged from 0.14 to 201 microg/g tattoo ink. Benz(a)pyrene was found in four ink samples at a mean concentration of 0.3 +/- 0.2 microg/g. We also found high concentrations of phenol ranging from 0.2 to 385 microg/g tattoo ink. PAHs partly show high quantum yields of singlet oxygen (Phi(Delta)) in the range from 0.18 to 0.85. We incubated keratinocytes with extracts of different inks. Subsequent UVA irradiation decreased the mitochondrial activity of cells when the extracts contained PAHs, which sufficiently absorb UVA and show simultaneously high Phi(Delta) value. Tattooing with black inks entails an injection of substantial amounts of phenol and PAHs into skin. Most of these PAHs are carcinogenic and may additionally generate deleterious singlet oxygen inside the dermis when skin is exposed to UVA (e.g. solar radiation).

Skin reactions are well described complications of tattooing, usually provoked by red inks. Chemical characterizations of these inks are usually based on limited subjects and techniques. This study aimed to determine the organic and inorganic composition of inks using X-ray fluorescence spectroscopy (XRF), X-ray absorption spectroscopy (XANES) and Raman spectroscopy, in a cohort of patients with cutaneous hypersensitivity reactions to tattoo. A retrospective multicenter study was performed, including 15 patients diagnosed with skin reactions to tattoos. Almost half of these patients developed skin reactions on black inks. XRF identified known allergenic metals - titanium, chromium, manganese, nickel and copper - in almost all cases. XANES spectroscopy distinguished zinc and iron present in ink from these elements in endogenous biomolecules. Raman spectroscopy showed the presence of both reported (azo pigments, quinacridone) and unreported (carbon black, phtalocyanine) putative organic sensitizer compounds, and also defined the phase in which Ti was engaged. To the best of the authors' knowledge, this paper reports the largest cohort of skin hypersensitivity reactions analyzed by multiple complementary techniques. With almost half the patients presenting skin reaction on black tattoo, the study suggests that black modern inks should also be considered to provoke skin reactions, probably because of the common association of carbon black with potential allergenic metals within these inks. Analysis of more skin reactions to tattoos is needed to identify the relevant chemical compounds and help render tattoo ink composition safer.

Tattooing has been an enduring form of body art since ancient times, but it carries inherent health risks, primarily due to the complex composition of tattoo inks. These inks consist of complex mixtures of various ingredients, including pigments, solvents, impurities and contaminants. This literature review aims to shed light on the organic and inorganic contaminants present in tattoo inks prior to the implementation of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation in 2022. This review shows that the most common contaminants are polycyclic aromatic hydrocarbons (PAHs), with a concentration range of 0.005–201 mg/kg, mainly detected in black tattoo inks, and primary aromatic amines (PAAs), with a concentration range of 0.5–1100 mg/kg, and heavy metals such as lead (0.01–14.0 mg/kg) and chromium(VI) (0.16–4.09 mg/kg) which are detected in almost all tattoo inks. When compared to the new concentration limits outlined in REACH, it is clear that a significant part of these contaminants would be considered non-compliant. However, the results of the review are limited due to the lack of quantitative data on contaminants in tattoo inks. In addition, the future implementation of REACH is expected to lead to changes in the composition of tattoo inks, which will affect the presence of contaminants.