Are Some Metals in Tattoo Inks Harmful to Health? An Analytical Approach (2024)

Tattoo application is widely performed all over the world; however,injection of coloring substances into the skin as metals may posea risk for allergies and other skin inflammations and systemic diseases.In this context, tattoo inks in green, black, and red colors of threebrands were purchased. Before starting the analysis, the acid mixturesuitable for microwave burning was determined, and according to theseresults, the inks were digested with nitric acid, hydrochloric acid,and hydrofluoric acid. Then, method validation was performed for tattooinks using inductively coupled plasma-mass spectrometry. The relativecontribution of metals to the tattoo ink composition was highly variablebetween colors and brands. Elements found in the main components ofinks are as follows (in mg kg^–1): Al, 1191.1–3424.9;Co, 0.04–1.07; Cu, 1.24–2523.4; Fe, 16.98–318.42;Ni, 0.63–17.53; and Zn, 2.6–46.9. It has been determinedby the Environmental Protection Agency that in some products, especiallythe copper element is above the determined limit. The analysis resultsobtained were classified by chemometric analysis, and the color andbrand relationship were determined. More toxicological studies arenecessary to understand the effects of tattoo inks containing heavymetals and/or organic components.

2.1. Reagents

All the acids used for digestionwere Supra-pure grade. Elemental calibration standards were preparedfrom 10 gmL^–1 of a multielement stock standard solution(Merck, Darmstadt, Germany). Tri-distilled ultrapure water was usedin all steps of the analysis, bi-distilled water was supplied by aMerck Millipore Milli 2 Integral 2 system (Molsheim France), and byusing classical distillation apparatus the third distillation wascarried out.

2.2. Apparatus

An inductively coupledplasma-mass spectrometer (ICP-MS 7800, Agilent, USA) was used forthe elemental analysis, and the operating conditions of the ICP-MSare given in Table 1.

2.3. Sampling

Three colors Red (r), Green(g), and Black (b) and three brands (A, B, C) of tattoo inks werepurchased from Turkish markets, and the color and the brands werechosen according to the popularity of the usage in Turkey. The inkswere in liquid form with different viscosity.

Ink samples weremicrowave-digested (Berghof Speedwave two, Eningen Germany), and aseries of studies were conducted to find out the best acid/acid mixturesfor degradation of the samples. Each combination was coded as givenin Table 2.

The precision was calculated on three replicates for all digestionprocedures, with optimum digestion condition being 0.3 g. The samplewas weighed in a Teflon vessel to which a mixture of 6 mL of HNO₃, 3 mL of HCl, and 0.8 mL of HF was added, and the microwaveoven was programmed 5 min 100 °C, 10 min 105 °C, and 75°C for cooling. The residue was dissolved in 5 mL of HNO₃ and, when necessary, the mixture was heated slowly to dissolvethe residue and filtered for ICP-MS analysis. The solution was transferredto a 10 mL volumetric flask and made up to volume.

2.4. Method Validation

The method wasvalidated by using A brand red tattoo inks. For the validation ofthe method, the accuracy, precision, limit of detection (LOD), limitof quantification (LOQ), linearity, range, sensitivity, and reproducibilitywere investigated.

The linearity of the method was determinedby measuring five different standard solution mixtures. Standard solutionswere prepared from 1000 μgg^–1 stock solutionof each element by dilution in different ranges. Each solution wasinjected three times.

The LOD is the lowest concentration of the analyte in a samplethat can be determined by an analytical method. The calculations weredone by analyzing blank solution. Measurements were made with themeasurement of blank samples. LODs and LOQs were experimentally calculatedas 3.3σ/S and 10σ/S,respectively, where σ is the standard deviation of the responseof 10 blanks and S is the slope of the calibrationcurve (EURACHEM 2000).

Sensitivity is the capacity of the test process to record smallchanges in concentration. It is the slope of the calibration curve.The regression line determined in the linearity section is sufficientto determine the sensitivity.

Range is the limit of an analytical method where the process betweenthe lowest and highest values is determined using the accuracy, linearity,and precision of a method by using standard solutions of each element.

Accuracy is defined as the proximity of the results to the realvalue. Accuracy of the method was determined with LGC7162-CRM (LGC,Germany). The systematic error was calculated, and then the t-test was performed to check the difference.

The precision of the method is the ability of the method to repeatany given value, or a degree of proximity of individual tests. Precisionwas evaluated in terms of intra and inter-run data distribution. Theintra-run precision represents the same day repeatability of the data,and inter-run represents different day repeatability. The precisionof the method was evaluated on standards, certified reference materials(CRMs), and real samples, and the results were given in RSD.

The blank level prior to measurement and instrumental drift werechecked during the validation. Temperature differences and some ofthe unexpected anomalies could cause drift. Standard solutions wereanalyzed after every 20 samples and at the end of the analytical procedure.The drift percentage was calculated by the formula: d% = C_n – C_iC_i × 100 where C_i is the concentration (μgL^–1) measured inthe standard solution immediately after the calibration curve and C_n is the actual concentration measured duringthe analytical sequence. A maximum d% of ±10%was considered acceptable for all elements.¹⁰

2.5. Chemometric Analysis

The JMP16 statisticalsoftware program was used for the chemometric analysis. The analyticalresults of the study were standardized in Excel 2016 and then importedto the JMP data table. ‘Data projection is performed mainlyby methods principal component analysis (PCA),’ Otto,¹¹ PCA was performed to the data, and the Scoreplot and Loading plot graphics were obtained for the chosen numberof components. The hierarchical cluster analysis was performed forgrouping the tattoos. The distances between the clusters are shownas a dendrogram. Dendrograms for cluster analysis are based on theWard method.

3.1. Digestion

In our study, indicatedelements (Al, Co, Cu, Fe, Ni, and Zn) have been detected in tattooinks. At first, the influence of acid digestion treatment on elementalanalysis was determined. For this purpose, six different mixturesof the acids were used as shown in Table 2. The mixture was selected based on referencesto strong acids used in tattoo inks, such as the study by Manso etal., 0.25 g of tattoo ink digested with 4 mL of nitric acid, 1 mLof hydrofluoric acid and 1 mL of hydrogen peroxide, and 0.2 g of inkdigested with nitric acid (65%, w/w) and hydrogen peroxide (30%, w/w).¹⁹

As shown in Table 3, solvent mixture IV was found to be moreappropriate for the sample preparation stage of real samples. A singleacid solution was also used for digestion, but results were undetectable.

3.2. Method Validation

Validation studiesare based on the ICH guideline (EMEA, Note for Guidance on Validationof Analytical Procedures: Text and Methodology, CPMP/ICH/381/95, 1995,1–15) within the scope of this study; Al, Co, Cu, Fe, Ni, andZn were analyzed in tattoo ink samples.

The lowest correlationcoefficient of all elements was 0.997. Standards were given to ICP-MSin the form of multielement analysis in accordance with its optimumrange. Linearity, range, sensitivity, LOD, and LOQ of the analysisare given in Table 4.

In our study, the precision parameter was obtained by standardsolutions and Cr tattoo inks. It is stated in the International Councilfor Harmonization (ICH) guideline that internal data are obtainedby reading each of the three concentrations covering a specific areathree times. The results are given in Table 5. It has been observed that the precisionof the method changes according to the element and the solution. Interdayprecision was lower than intraday precision, as expected.

Accuracy of the method can be checked for Co, Ni, and Zn. Systematicerror was calculated and then the t-test was applied.There is no significant difference between the CRM and sample (p ≤ 0.05).

Microwave-digested tattoo ink samples were analyzed in ICP-MS usingan internal standard after appropriate dilutions. The results aregiven in Table 6.

There are limited studies regarding the element content of tattoos,especially in Turkey. Piccinini et al. published the JRC report5 andreported Co, Cu, and Zn values as 6.8, 31.8, and 21.6%, respectivelyin tattoo inks.²⁰ The comparison of thereferences about elemental analysis in tattoo inks is given in Table 7. In this study, theamount of elements in tattoo inks showed great variations among somecolors and some brands. This is particularly notable for the elementCu.

The amount of Cu in green in inks was higher in all three brands.The reason why the amount of Cu in green inks is higher than thatof red and black ink tattoos is thought to be related to the colorfactor and the amount of elements it contains in tattoo inks. Whilethe amount of Cu is higher in green and red colored tattoo inks inB brand, this amount was higher in A brand in black colored inks.Cu is a metal with the capacity to initiate oxidative damage in cellsand is thought to induce cellular toxicity.²¹ The limit value for Cu was determined as 25 mg kg^–1. It is seen that this limit is exceeded in the analyzed samples,especially in green inks (213.6–2523.4 mg kg^–1).

Cu is an essential mineral. Data on dermal toxicity caused by Cucompounds are insufficient. It is reported in EC regulation 1272/2008that CuSO4 is skin irritant 2 ((ResAP 2008)1).⁹

Li et al., in their study, determined the irritating effect ofCu on the skin. However, copper-peptide (GHK-Cu) has low potentialto cause skin irritation and therefore offers a safer alternativeto the transdermal delivery of copper.²² There are also some papers related to the contact dermatitis effectof Cu.^23,24

Al concentration was determined in the range of 1191.1–3424.9mg kg^–1 in all analyzed samples, and it is quitehigh compared to all other element amounts. Al content was found tobe close to each other in all color inks of B and C brands. Al hasbeen found in components of some inks as cobalt aluminate. Al saltsare used in red and purple inks. In a study of 30 tattoo inks, 87%reported the presence of Al.²⁵ Co and Alare known to cause granulomatous reactions^26,27 It is reported in EC Regulation 1272/2008 that AlCl₃ isskin corrosive 1B ((ResAP 2008)1).⁹ Exposureof the skin to low doses of aluminum chloride for 18 weeks has beenshown to result in aluminum accumulation in the brain.²⁸ Moreover, it has been observed that intradermalinjection of aluminum salts increases granuloma formation. Zn hasan irritating effect on the skin as well.^28,29 Al skin penetration is insignificant in healthy individuals, butimportant in shaved adults. While the injected Al dose is desiredto be 25 g L^–1, the maximum parenteral dose notto exceed 1 mg kg^–1 day so that Al does not accumulatein the blood circulation.³⁰

Zn is an essential element for many intracellular molecular reactionsand may play an important role in the induction of apoptosis. It ishypothesized that apoptosis induces the toxicity of cadmium throughits interaction with the Zn finger protein. When the Zn contents ofdifferent brands and colors were evaluated, it was determined thatAr ink had the highest Zn content (46.90 mg kg^–1). Today, ZnO is used as a UV filter in sunscreens, as well as increams to relieve skin damage such as burns, wounds, and irritations.It is reported that the use of this compound is safe. Zinc chlorideis listed as an inactive ingredient in FDA-approved drug productsto be administered subcutaneously (0.006%) and intradermally (0.7%).³¹ In EC Regulation 1272/2008 ZnCl₂ isreported to be corrosive to skin ((ResAP 2008)1).⁹

In tattoo inks, Fe forms red (Fe₂O₃), black(Fe₃O₄), yellow (FeOOH), and brown (iron oxidemixture) colors in different formulas. Iron oxide is a known darkenerused in tattoo inks. These iron oxides are present in inorganic inks,albeit in small quantities. It is reported that it reacts with O₂ and H₂O and turns into different salts. Iron oxideformation has been associated with significant deleterious effects,such as inflammation, apoptosis, disruption of mitochondrial function,membrane changes, reactive oxygen species formation, increased micronucleusinduction, and chromosome condensation, depending on concentration,exposure time, and cell type.³²

It was emphasized that iron accumulation due to iron oxide compoundscaused a decrease in the GSH level in neural tissues and inductionof oxidative stress. In addition, it has been reported that iron oxide-basedpigments can react during magnetic resonance imaging scans and triggerlow-grade burns³³ Dixon et al. reportedthat iron accumulation causes an increase in cytotoxic lipid oxidationin the cell.³⁴ Likewise Imam et al. reportedthat iron oxide nanoparticles cause damage to the membrane of ratbrain endothelial cells by producing ROS.³⁵

It is also reported that iron oxide pigments always contain a smallamount of Ni as an impurity. It is interpreted that Ni may cause allergicreactions. Fe concentration was determined in the range of 16.98–318.42mg kg^–1 in all analyzed samples in this study. Thehighest Ni concentration was found in the Ar sample as 17.53 mg kg^–1. ResAP(2003)2 and ResAP(2008)1 define the limit valuefor Ni as ‘as low as technically achievable’.^8,9 Ni is an immunotoxic, neurotoxic, and carcinogenic agent. Dependingon the dose and exposure time, it can cause a variety of effects,including contact dermatitis, cardiovascular diseases, asthma, lungfibrosis, and respiratory tract cancer. Ni causes oxidative stressand mitochondrial dysfunctions. Chronic exposure causes accumulationof nickel and nickel compounds in the body. Nickel²⁺ exposurehas been associated with DNA hypermethylation and transcriptionalrepression of tumor suppressor genes in vitro and in vivo. It hasbeen reported that nickel ions trigger apoptosis by acting on caspasesin the cell. Intradermal nickel exposure can cause scaly red areasand localized erythematous, pruritic vesicles.^36,37

Cobalt is an essential element required for vitamin B12 synthesisin the body. However, high levels cause adverse effects. Cobalt hasno place in the diet. It is taken 5–40 μg daily withnutrition. Its total level in the body is estimated to be between1.1 and 1.5 mg.³⁸ It causes adverse effectswhen taken in high doses. Cobalt can cause allergic contact dermatitis,eye irritation, and prolonged contact sensitization.³⁹ The International Agency for Research on Cancer has listedcobalt and cobalt compounds as agents that are possibly carcinogenicto humans (Group 2B). A study in rabbits exposed to cobalt chlorideat a dose of 1354 μg/mL for 18 days reported marked neuropathywith demyelination and atrophy of the optic nerves.³⁸ It has been reported that cobalt ions cause oxidative stressand cytotoxicity by generating a large number of reactive oxygen speciesand HO radicals in cells.⁴⁰ Cobalt ionsand cobalt nanoparticles were found to be effective on cell signals,enzymes, and cell metabolism. It has also been shown to interact withvarious receptors, ion channels, and biomolecules in cells.^40,41 It has been reported that cobalt ions can replace essential metalions by interacting with metal-based proteins in the cell (e.g., Mg²⁺, Ca²⁺, Zn²⁺, etc.), thus causing dysfunctionin these enzymes or proteins.⁴² All thesefindings support the idea that cobalt ions and nanoparticles can causecell death due to oxidative stress. There is no FDA-specified limitvalue for cobalt in tattoo inks (ResAP(2003)2 and ResAP(2008)1). Coconcentration was determined in the range of 0.04–1.07 mg kg^–1 in all analyzed samples in this study.

In 2009, Forte et al. conducted a survey with tattoo inks of differentbrands and colors.⁴ One of the brands thatwere used in this study is the same brand that is used in our study.When the data from the two studies were compared, it was seen thatthe amounts we obtained in general were higher.⁴³

Lim and Shin analyzed Al, Co, Cu, Fe, Ni, and Zn in tattoo inkswithout classifying the color. The results were 0.1, 1.7, 1840, 24,700,5, and 8.7 mg kg^–1, respectively. Cu, Fe, and Nivalues were higher than those in this study.⁴⁴

In the study by Arl et al., high concentrations of Al and Cu levelswere identified in green inks in tattoo inks, although not specifiedon the labels. Cu and Fe are commonly applied in small amounts inblack and red inks.⁴⁵

In Turkey, there are few studies on this topic. Kılıçet al. worked toxic metals in some cosmetic products consumed in Turkeyand they found a concentration of Co 0.1 and 0.9, Cu 0.3 and 2.2,and Ni 0.3, 2.5, and 3.3 mg kg^–1.⁴⁶ In another study, Co, Cu, and Ni were determined in fingerpaint samples in Turkey, and, for red color paint Ni concentrationwas found to be 1.5 mg kg^–1 and in green paint Cuand Ni concentrations were found to be 0.5 and 2.3 mg kg^–1, respectively. The other elements could not be detected in thisstudy.⁴⁷ The amounts of the elements varyaccording to the type and color of the material.

Some studies were done in biopsies from tattooed skin samples tofind the effect of some elements coming from tattoos on skin. Serupet al. analyzed Cu, Fe, and Ni in both tattoo inks and biopsies.⁷ They found 1608.7, 2317.8, and 0.7 and 42.93,3.48, and 1.05 mg kg^–1, respectively. They had higherresults with tattoo inks overall. De Cuyper et al. found 4.3, 5.9,21, and 0.4 mg kg^–1 of Al, Cu, Fe, and Ni, respectively,in biopsies taken from tattooed skin. As can be seen, the resultsin biopsy samples are different from each other.⁴⁸ Considering all these evaluations, it can be said thatthe amount of trace element varies depending on the color and brand.

3.3. Chemometric Analysis

Multivariateanalysis was performed for the classification of the tattoo inks accordingto their elemental pattern. The tattoo data in Figure ​Figure11 show that 57.6% of data variance can beexplained by use of two principal components (Figure ​Figure11). PCA on the basis of correlation matrixof the data provides the results given in Figure ​Figure11 for the scores and loadings. It has beenpossible to group the different brand of tattoos according to theircolor by the score plot. The loading plot provides the projectionof the features on the principal components. There is the greatestcorrelation for the elements Zn and Co, both on the same line. Thefeatures have correlation except Cu.

The dendrogram obtained by cluster analysis using the Ward methodfor raw ICP-MS data (Figure ​Figure22) distinguished the data. It can be said there are two maingroups in the data. Ag, Ab, Cr, Bb, Cb, and Ar are in one group andBg, Cg, and Br in the second group. Bb and Cb have highest correlationand Ar is the worst.

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