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Diss Factsheets

Toxicological information

Direct observations: clinical cases, poisoning incidents and other

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Administrative data

Endpoint:
direct observations: clinical cases, poisoning incidents and other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not specified
Reliability:
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: Any kind of reliability rating is not considered to be applicable, since human studies/reports are not conducted/reported according to standardised guidelines.

Data source

Reference
Reference Type:
publication
Title:
Synchrotron-based ν-XRF mapping and μ-FTIR microscopy enable to look into the fate and effects of tattoo pigments in human skin
Author:
Schreiver, I. et al.
Year:
2017
Bibliographic source:
Scientific Reports 7: 11395.

Materials and methods

Study type:
study with volunteers
Endpoint addressed:
basic toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the present study, tattooed human skin and regional lymph nodes originating from four donors (corpses) were analysed. Inductively coupled plasma mass spectrometry was used to assess the general contents of elements in both tissues and tattoo inks after microwave digestion. Laser-desorption/ionization time-of-flight MS facilitated the identification of organic pigments in enzyme-lysed samples. To precisely locate the different elements in the cutaneous and lymphatic microenvironments, to provide information on TiO2 speciation and to assess primary particle sizes at the nanometric scale in particle mixtures, synchrotron-based X-ray fluorescence investigations have been performed at both the micro (μ-XRF) and nano (ν-XRF) range. Furthermore, biomolecular changes in the respective tissues were assessed at the micrometric scale and in the proximity of tattoo particles using synchrotron-based Fourier transform infrared (μ-FTIR) spectroscopy.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Titanium dioxide
EC Number:
236-675-5
EC Name:
Titanium dioxide
Cas Number:
13463-67-7
Molecular formula:
O2Ti
IUPAC Name:
dioxotitanium
Test material form:
not specified
Details on test material:
not specified
Specific details on test material used for the study:
not specified

Method

Type of population:
general
Subjects:
- Number of subjects: 4 donors with tattoos / 2 donors without any tattoos
- Demographic information: all samples were obtained anonymously without information on the patients demographics.
- Known diseases: all samples were obtained anonymously without information on the patients disease status or cause of death.
Ethical approval:
other: ethical approval of human biopsy samples was granted by the Ethics committee of the Medical Faculty of the Ludwig-Maximilians University of Munich
Route of exposure:
dermal
Reason of exposure:
intentional
Exposure assessment:
not specified
Details on exposure:
Tissue samples of four individuals tattooed with orange, red, green or black and two non-tattooed control donors were analyzed for the presence of organic pigments. The sample size was limited by the availability of specimens and the beamtime.
Examinations:
HUMAN PREPARATION
Samples of tattooed skin and regional lymph nodes as well as skin and lymph node samples of donors without any tattoos were taken postmortem at the Institute of Forensic Medicine at the Ludwig-Maximilians University of Munich (court-ordered autopsies without any additional cosmetic impairment to the skin). The experiments were performed according to the Helsinki Declaration of 1975.

Tissue samples were stored at −20 °C directly after excision and further processed for analysis within a year. Subsamples were cut and frozen in TissueTek O.C.T. matrix for cryo-microtome sectioning. Sections of 5 or 6 μm were obtained and mounted on BaF2 substrates for μ-FTIR and μ-XRF measurements at ID21. Sections for fluorescence light microscopy had a thickness of 6 – 10 μm and were deposited on standard glass slides, while ν-XRF analyses at ID16B were performed on 12 – 14 μm sections on 4 μm Ultralene window films mounted on Si3N4 windows. Sections were inactivated using 4% formaldehyde buffer for 10 minutes and subsequently washed with deionized water. For μ-FTIR and μ-XRF analyses, samples were freeze-dried and stored in a dehydrated environment. Sections on microscopic glass slides were mounted in DAPI-Fluoromount G for cell nucleus staining.

ICP-MS ANALYSIS
Elemental compositions of in total 20 skin and 25 lymph node samples of tattooed donors as well as 2 skin and 2 lymph node samples of non-tattooed donors were analyzed using a nitric acid microwave digestion. Samples were directly adjacent to those used in other parts in this investigation. Elemental concentrations given in ppm are calculated in relation to the weight of digested tissue. Standards for ICP were Sc, Al, Cu, Ni, Hg, In, Cr, Fe and Cd.
A 20-fold dilution of each sample was prepared including 10 ppb of the elements In and Sc as internal standards. XSeries II ICP-MS together with an ESI SC2 autosampler were used for sample analysis. Sample analysis was carried out in triplicate with 100 sweeps each. Resolution was set to 0.02 amu and the dwell time for all elements was 10 ms. Measurements were carried out with collision cell in either −3.0 V mode (In, Sc, Cr, Fe, Ni, Cu, Cd) or 0.0 V mode (Sc, Al). H2/He (7% v/v) was used as the collision gas with 5 ml/min flow rate.

LDI-ToF-MS IDENTIFICATION OF ORGANIC PIGMENTS
In total 8 skin and 8 lymph node samples of tattooed donors as well as 2 skin and 2 lymph node samples of non-tattooed donors were analyzed. Samples were lysed using collagenase from Clostridium histolyticum Type IA with an incubation time of at least 24 hours at 37 °C. Lysates were heat-inactivated at 90–95 °C for at least 12 hours. Precipitated pigment particles were washed twice with PBS. Centrifugation was carried out and precipitates were applied to a ground steel target plate and measured using an UltrafleXtreme MALDI-ToF/ToF. Spectra were obtained by averaging 3000 individual spectra, with a laser rate of 1000 Hz in positive reflector mode. The instrument was calibrated prior to each measurement with an external ProteoMass™ MALDI Calibration Kit. Data were processed using the flexControl 3.4 and flexAnalysis 3.4 software.

SYNCHROTRON FTIR MICROSCOPY
FTIR microscopy analyses were performed at beamline ID21 (Susini et al., 2007)*. The beamline is equipped with a Thermo Nicolet Continuum microscope coupled to a Thermo Nicolet Nexus FTIR spectrometer with a 32x objective, a motorized sample stage, and a liquid nitrogen-cooled 50 μm HgCdTe detector. Maps were acquired in transmission mode using a 10 × 10 μm² beam, step size of 8 μm. Spectra were recorded as an average of 64 scans per spectrum, over a range of 4000 to 850 cm−1 and with a spectral resolution of 4 cm−1. The OMNIC software was used to transform spectra from maps of skin and lymph node samples to second derivatives using Savitsky-Golay of second polynomial order with 21 smoothing points (Martin et al., 2010; Benseny-Cases et al., 2014). Unscrambler X software (Version 10.3) was used for further statistical analysis. Principal component analysis (PCA) was performed on the mean-centered data using the spectral regions from: 1800 to 1350 cm−1 (related to proteins) and 3200 to 2800 cm−1 (related to lipids)(Chwiej et al., 2010; Pettbots et al., 2007). PCA was performed separately for skin and lymph node samples. Score plots and loading plots obtained by PCA analysis as well as mean values from the regions of interest were used for data interpretation.

SYNCHROTRON μ-XRF AND μ-XANES.
μ-XRF and μ-XANES analyses were carried out at the beamline ID21 (Salomé et al., 2013)*. X-rays were generated by an U42 undulator operated in “gap-tracking” mode, i.e. the gap value was optimized for each energy. A fixed exit double-crystal Si(111) Kohzu-monochromator was used in combination with a Ni-coated flat double-mirror rejecting high-energy harmonics and allowed for energy selection with about 0.4 eV resolution of the primary radiation at Ti K-edge (5.1 keV). Downstream of the monochromator, the beam was focused down to 0.4 × 0.8 μm2 (vertical × horizontal) using a fixed-curvature Kirkpatrick-Baez (KB) mirror system. The flux was 1.6 × 10^10 photons/s (~180 mA SR current in multi-bunch mode). A 30 μm Al attenuator was used to reduce the photon flux by one order of magnitude to keep the XRF detector dead time within its linear range. A photodiode collecting the XRF from a thin Si3N4 membrane inserted in the beam path was used to continuously monitor the incoming beam intensity. XRF and scattered radiation were collected with a dispersive energy silicon drift detector with an active area of 80 mm². Acquisition time per point was 100 ms. The pixel size for collecting the XRF maps was adjusted to the regions of interest and varied from 0.5 μm to 5 μm. Scans were performed in continuous (zap) mode and an energy of 5.05 keV was selected for μ-XRF mapping. For collecting Ti XANES spectra, the energy of the incoming beam was scanned from 4.95 to 5.1 keV in increments of 0.5 eV, with acquisition times of 100 ms per energy. Depending on the concentration of the probed region, between 1 and 10 μ-XANES spectra were collected per point and subsequently averaged. Full-field XANES maps were also collected to total the XANES spectra over multiple pixels.

SYNCHROTON ν-XRF.
The analysis on an adjacent section of skin and lymph node tissue from donor 4 was performed by means of ν-XRF at ID16B. The experimental set-up is described elsewhere(Martinez-Criado et al., 2016)*. A pink beam with an energy of 17.5 keV with ΔE/E = 1% was focused down to 50 × 50 nm² using KB mirrors. The flux of >1 × 1011 photons/s was subsequently reduced using gold and silicon attenuators to keep the dead time on the XRF detectors within the linear range. Two three-element silicon drift detector arrays were used. The two ν-XRF maps were recorded with a step size of 50 × 50 nm² and 100 ms dwell time. In contrast to the set-up installed at ID21, ID16B operates in air. For estimating the particle size of TiO2, analysis was performed on 10 particles by computing the full width at half maximum of line profiles through the particles.

*References
- Susini, J. M. et al. Technical Report: The FTIR Spectro-Microscopy End-Station at the ESRF-ID21 Beamline. Synchrotron Radiat. News 20, 13–16 (2007).
- Martin, F. L. et al. Distinguishing cell types or populations based on the computational analysis of their infrared spectra. Nat. Protocols 5, 1748–1760 (2010).
- Benseny-Cases, N., Klementieva, O., Cotte, M., Ferrer, I. & Cladera, J. Microspectroscopy (μFTIR) reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains. Anal. Chem. 86, 12047–12054 (2014).
- Chwiej, J. et al. Synchrotron FTIR micro-spectroscopy study of the rat hippocampal formation after pilocarpine-evoked seizures. J. Chem. Neuroanat. 40, 140–147 (2010).
- Petibois, C., Drogat, B., Bikfalvi, A., Deleris, G. & Moenner, M. Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging. FEBS Lett. 581, 5469–5474 (2007).
- Salomé, M. et al. The ID21 Scanning X-ray Microscope at ESRF. J. Phys. Conf. Ser. 425, 182004 (2013).
- Martinez-Criado, G. et al. ID16B: a hard X-ray nanoprobe beamline at the ESRF for nano-analysis. J. Synchrotron Radiat. 23, 344–352 (2016).
Medical treatment:
not applicable

Results and discussion

Clinical signs:
not specified
Results of examinations:
ORGANIC PIGMENTS TRANSLOCATE FROM SKIN TO LYMPH NODES
- detection of the same pigment species in both skin and regional lymph nodes revealed the drainage of tattoo particles in two out of four tattooed donors (donors 3 and 4)
- for the two donors (donors 1 and 2), the absence of organic pigments in the lymph nodes suggests either concentrations below the limit of detection (approx. 0.1–1% w/w pigment per extract), possible metabolic decomposition or drainage to alternative lymph nodes.
- in one donor (donor 2), in which no organic pigments were found in both skin and lymph node, the general ability for azo pigment translocation to lymph nodes was proven in additional skin and lymph node samples.
- colour-giving pigments in lysed tissues were found to be copper phthalocyanines with either hydrogen, chlorine or bromine residues in three out of four skin samples. Reddish parts of the tattoos contained the azo group-containing pigments red 170 and orange 13.
- no xenobiotic pigment particles were detected in either skin or lymph tissue of the control samples.

TATTOOS CONTRIBUTE TO THE ELEMENTAL LOAD OF LYMPH NODES
- the experimenters found Al, Cr, Fe, Ni and Cu quantitatively elevated in skin and lymph node specimens using ICP-MS analysis.
- for one donor (donor 4), Cd and Hg concentrations were found increased only in the lymph nodes, but not in the analyzed skin sections (elements probably result from other tattoos that were not part of this study or other routes of exposure drained through the same lymphatic tissue).
- non-quantitative evaluation of the survey scans revealed the presence of titanium, presumably derived from TiO2, in all tattooed skin samples but not in controls.
- elevated levels of Fe found in the skin and lymph nodes of donor 4 imply an additional use of iron-based pigments.
- in donors 1, 2 and 3, Fe concentrations were only increased in adjacent lymph nodes and not in the corresponding skin samples.
- in donor 4, the use of pigment copper phthalocyanine green 36, as identified with LDI-ToF-MS, is reflected by high amounts of Cu in skin and lymph nodes as well as the non-quantitative detection of Br.
- pigment copper phthalocyanine green 7 was well detectable with our LDI-ToF-MS approach in the skin of donor 2, but it was not detectable in the corresponding regional lymph node. Increased Cu levels measured by ICP-MS in this adjacent sample, suggest the presence of this copper phthalocyanine pigment.
- in donor 2, Cu levels in lymph nodes are strongly increased despite the fact that green 7 could not be detected with LDI-ToF-MS. Adjacent samples of tissue were used for each analysis. - given the nature of the samples, pigment deposition within skin and lymph nodes is not homogeneous and therefore explaining the different findings.
- non-tattooed control donor 1 had slightly elevated levels of about 13 ppm Cu in the lymph nodes (in the range of the average 5.89 ± 8.03 ppm of Cu detectable in lymph nodes of female cadavers (Saltzman et al., 1992)*).
- Ni and Cr were found in the human specimens.
- Ni levels were increased in the skin and lymph nodes of donor 2 and 3 (likely source: tattoo).
- Ni concentrations of 0.28–10.05 ppm total tissue weight found here are within the range of 0.8–3.7 ppm dry weight Ni in hilar lymph nodes (Rezuke et al., 1987))*.
- Cd was drastically elevated only in the lymph node of donor 4.
- for all other samples, Cd tissue burdens lie within normal values (Saltzman et al., 1992)*.
- Al was present in skin and lymph node tissues of the three tattooed donors 2, 3 and 4. Since auxiliary lymph nodes have been investigated in the case of donor 2 and 3, co-exposure from antiperspirants containing various aluminum salts cannot be excluded, neither in tattooed nor control samples.
- Al concentrations in the controls were lower.
Please also refer to table 1 in the field "Any other information on results incl. tables" below.

μ-XRF MAPPING LINKS METALLIC ELEMENTS TO TATTOO PARTICLES
- the majority of particles in the skin tissue were surrounded by phosphor-rich nuclei visualized by DAPI staining in fluorescence light microscopy and integration of the element P in μ-XRF analysis
- LDI-ToF-MS analysis revealed the presence of pigment green 36 and the following ν-XRF results from ID16B acquired at 17.5 keV, i.e. above Br K-edge (13.47 keV) undermined the primarily Br-related contribution.
- tattoo particles containing Ti and Br are adjacent to each other with only a slight overlap in skin and seem to be more evenly co-localized in lymph tissue. Both elements were found in the dermis of donor 4 directly beneath the cell nuclei-rich epidermis and up to a few hundred micrometers deep in the skin. In the lymph nodes, some particles were deposited in the stroma directly beneath the capsule. The bulk of Ti and Br containing particles, however, became visible as pigment agglomerates at a distance of about 250 μm to the lymph node capsule.
- Cl concentrations are highest in the lymph node capsule and lower concentrations can be found in the particle region as part of the pigment phthalocyanine green 36.
- all analyzed samples from the tattooed donors contained Ti.
- other highly abundant elements are K and Ca as they are physiologically present in cells.
- micro X-ray absorption near edge structure (μ-XANES) spectra at the Ti K-edge were collected for the skin and lymph nodes of donors 1, 3 and 4. The spectra of donor 4 showed more qualitative correlation with the reference spectrum of rutile than with that of anatase. A clear switch of peak maxima between 4.99–5 keV occurs as a difference of both types of crystal structures. A calculated spectrum of 20% anatase and 80% rutile mixture is not clearly distinguishable from pure rutile, but shows a pre-edge at around 4.97 keV, similar to the μ-XANES spectra of the tattooed samples. Therefore, mostly rutile TiO2 is present in all tattooed donors, with minor amounts of anatase.

PARTICLE SIZE VARIES BETWEEN PIGMENT SPECIES
- μ-XRF maps of skin and lymph node sections show large tattoo particle agglomerates up to several micrometers.
- donor 4 (ν-XRF): three different pigment particles were detected, each showing a different elemental composition and distribution within the same area. The average particle size of TiO2 in both skin and lymph nodes was 180 nm with a standard deviation of 23 nm and a standard error of 7 nm. This rather large particle size does not prevent distribution via the lymph fluid.
- pigment phthalocyanine green 36 analyzed by ν-XRF mapping of Br was much more polydisperse, with particles presumably smaller than the resolution of 50 nm and up to the μm range in skin. In lymph node tissue, particles containing Br were smaller, with fewer particles of a larger size.
- skin and lymph node of donor 4 contained Cu, related to the identified copper phthalocyanine pigments, and its maps show perfect co-localization with Br.
- Fe particles were present in the lymph node but not skin tissue and therefore possibly originate from another tattoo or route of exposure.

TATTOO PARTICLES INDUCE BIOMOLECULAR CHANGES
- donor 4: dermis without pigment and dermis around pigment particles have fewer lipid-related long alkyl chains (–CH2 stretching mode, asym. at 2920 cm−1 and –CH2 sym. at 2854 cm−1) and ester (–C = O stretching mode, peak at 1745 cm−1) vibrations than epidermis, and that dermis around pigment particles regions contain higher levels of lipids than dermis without pigment. Dermis around pigment particles can be separated from dermis without pigment and epidermis since the latter two have higher protein concentrations.
- donor 4: in the proximity of the pigment particles, the protein content is lower compared to other parts of the collagen-rich dermis. Crosslinkred collagen fibers becomes more pronounced close to the particles. Lipid content are also higher in the proximity of particles compared to other parts of the dermis.
- in the skin of donor 2, a similarly enhanced lipid content and the presence ofcrosslinked collagen fibers in the dermis around particles were noticed.
- a statistical comparison of particle-containing and particle-free areas in the lymph node tissue of donor 4 showed a similar increase of lipid contents in the former. No consistent differences in the kind of protein folding could be observed in lymph nodes.

*Reference
- Saltzman, B. E., Gross, S. B., Yeager, D. W., Meiners, B. G. & Gartside, P. S. Total body burdens and tissue concentrations of lead, cadmium, copper, zinc, and ash in 55 human cadavers. Environ. Res. 52, 126–145 (1990).
- Rezuke, W. N., Knight, J. A. & Sunderman, F. W. Reference values for nickel concentrations in human tissues and bile. Am. J. Ind. Med. 11, 419–426 (1987).
Effectivity of medical treatment:
not specified
Outcome of incidence:
not specified

Any other information on results incl. tables

Table 1: Element concentrations per tissue weight (ppm) in human skin and lymph node samples analyzed by ICP-MS. Abbreviations: LN = lymph node. Elements measured (non-specified oxidation states): aluminum (Al), barium (Ba), bromine (Br), cadmium (Cd), chromium (Cr), copper (Cu), iodine (I), iron (Fe), lead (Pd), manganese (Mn), mercury (Hg), nickel (Ni), rubidium (Rb), titanium (Ti), tungsten (W), and zinc (Zn). #Non-quantitatively identified elements (elements marked in bold are associated with tattoo pigments). a: Wet basis, average from 21 male/female cadavers (Saltzman et al., 1990)*. b: Tissue dry weight, average in hilar lymph nodes from 3 cadavers (Rezuke et al., 1987)**. cTissue dry weight, average in hilar lymph nodes from 12 male cadavers (Teraoke, 1981)***.

Donor

Tissue

Location

Al

Cr

Fe

Ni

Cu

Cd

other#

1

Skin

Dorsal

0.92

0.74

64.7

0.59

2.51

0.15

Ti

 

LN

Left axillary

1.97

0.43

125

0.28

2.98

0.35

Zn, Rb

2

Skin

Right leg

7.29

5.54

51.1

2.51

18.8

0.23

Ti

 

LN

Right inguinal

9.06

22.5

235

10.1

118

1.23

Ti, Mn

3

Skin

Right arm

3.39

2.73

84.7

1.61

67.5

0.17

Ti, I

 

LN

Right axillary

5.08

13.9

221

6.74

28.7

0.28

Ti, Mn, Zn,

Rb, I

4

Skin

Left arm

15.4

4.07

120

0.45

199

0.52

Ti,Br

 

LN

Left axillary

4.16

0.67

138

0.30

15.3

146

Ti,Br,Ba,

Mn, W, Rb,

Hg

Control 1

Skin

Proximal

0.75

0.16

35.6

0.08

1.44

0.12

Pb

 

LN

Axillary

1.11

0.31

64.4

1.09

12.9

0.47

 

Control 2

Skin

Proximal

0.76

0.60

37.6

0.15

1.41

0.25

 

 

LN

Axillary

0.24

0.14

74.7

0.09

2.48

0.83

Zn, Rb

Literature values

Skin

 

 

 

 

 

0.35/0.42a

0.05/0.02a

 

 

LN

 

2000c

8.2c

1800c

0.28b 3.7c

2.94/ 5.89a 7.6c

0.24/ 0.20a 2.5c

 

* Saltzman, B. E., Gross, S. B., Yeager, D. W., Meiners, B. G. & Gartside, P. S. Total body burdens and tissue concentrations of lead, cadmium, copper, zinc, and ash in 55 human cadavers. Environ. Res. 52, 126–145 (1990).

** Rezuke, W. N., Knight, J. A. & Sunderman, F. W. Reference values for nickel concentrations in human tissues and bile. Am. J. Ind. Med. 11, 419–426 (1987).

*** Teraoka, H. Distribution of 24 elements in the internal organs of normal males and the metallic workers in Japan. Arch. Environ. Health 36, 155–165 (1981).

Applicant's summary and conclusion

Conclusions:
According to the authors, they found a broad range of tattoo pigment particles with up to several micrometers in size in human skin but only smaller (nano)particles transported to the lymph nodes. With the detection of the same organic pigments and inorganic TiO2 in skin and lymph nodes, they mentioned that these findings provide strong analytical evidence for the migration of pigments from the skin towards regional lymph nodes in humans. The authors also stated that they were able to prove the presence of several toxic elements, such as Cr and Ni, derived from tattooing. However, the experimenters stated that elemental deposits in lymph nodes which were not found in the corresponding skin revealed that tattooing might not have been the only route of exposure in these particular individuals whose tissues were removed after their demise.