# Modulation of the Toxicity of Three Red Tattoo Pigments by Artificial Aging

Lise Aubry, Marie Blanchemanche, Lucie Breuillé, Thierry Rabilloud, Thierry Douki

► To cite this version:

Lise Aubry, Marie Blanchemanche, Lucie Breuillé, Thierry Rabilloud, Thierry Douki. Modulation of the Toxicity of Three Red Tattoo Pigments by Artificial Aging. Chemical Research in Toxicology, In press, 10.1021/acs.chemrestox.5c00451 . hal-05522874

HAL Id: hal-05522874

https://hal.science/hal-05522874v1

Submitted on 23 Feb 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Modulation of the toxicity of three red tattoo
pigments by artificial aging

Lise Aubry†, Marie Blanchemanche†, Lucie Breuillé†, Thierry Rabilloud‡, and Thierry Douki†,‡

† Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG, SyMMES, F-38000 Grenoble,
France

‡ Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG
LCBM, F 38054 Grenoble, France

* Corresponding author:

Thierry Douki

SyMMES UMR 5819, CEA – UGA - CNRS - Grenoble-INP, DRF/IRIG, CEA-Grenoble, 17

Avenue des Martyrs, 38054 Grenoble Cedex 9, France; [orcid.org/0000-0002-5022-071X](https://orcid.org/0000-0002-5022-071X)

Email: thierry.douki@cea.fr
ABSTRACT. Pigments used in tattoos represent a unique case of voluntary long-term exposure to particles trapped in the skin. Evidence is accumulating that these compounds, and in particular red pigments, are associated with skin disorders and possibly cutaneous cancers. In the present work, we tested the hypothesis of a degradation of pigments in the dermis that would lead to the release of diffusible nanoparticles or soluble degradation products. These species could then reach the epidermis and trigger physiological responses by impacting keratinocytes. Two degradation pathways of tattoo pigments were investigated in the present work that combines physico-chemical characterization and toxicological assessment in the HaCaT human keratinocyte cell line. The first pathway was the photodegradation of pigments, which we explored in a previous work on Pigment orange 13 (PO13) and extended here to Pigment red 254 (PR254) and Pigment red 122 (PR122). While PR254 was photostable, PR122 was found to release toxic photoproducts. The second studied pathway was the possible degradation of in the phagolysosomes of macrophages where pigment particles are stored in the skin. We did not observe degradation upon incubation in reconstructed phagolysosomal medium, but rather found that the added immunoglobulins completely inhibited the cytotoxicity of PO13, PR254 and PR122. The observation of a drastic decrease in Zeta potential strongly suggested the creation of a protein corona which led to a decrease in cellular toxicity.

KEYWORDS

Tattoo; pigments, photodegradation, phagolysosomes, toxicity
GRAPHICAL ABSTRACT

<figure><img src="image_2.png" /></figure>
# INTRODUCTION

Tattooing is a long-time practice in many cultures and specific groups in societies. In recent years, it has spread into the general population in industrialized countries. In 2019, the prevalence of tattoos ranged from 11.7 to 31.5% in the overall population depending on the country ¹. More recent data report a value of 32% for the USA, with larger prevalence in the 18-26 and 30-49 age groups ². The tattoo industry ³ advertises even larger prevalence values, as large as 46% for the USA and more than 40% for most European countries with Italy and Sweden as the most concerned countries (48 and 47%, respectively) ³. A large fraction of tattooed people has more than one tattoo ¹, ² and women are more tattooed than men. Tattoos are predominantly black but red is the second most used color followed by blue and green ⁴. The rising popularity of tattooing raises some concerns regarding cutaneous health ⁵, ⁶. Allergic reactions and photosensitivity issues have been reported ⁷⁻⁹. The particular feature of these reactions is that they could occur several years after tattooing ⁸, ¹⁰. Some concerns have also been raised on the link between skin cancer and tattoos ¹¹⁻¹⁶, although no strong animal ¹⁴ or epidemiological evidence is currently available ¹⁵, ¹⁷, ¹⁸.

From a toxicological point of view, tattooing represents a unique situation of voluntary long-term exposure to artificial compounds which can only be removed by drastic procedures such as laser ablation ¹⁹, ²⁰. Tattooing consists in the injection of ink into the dermis. Inks contain many different components including water, solvents, preservatives, fillers and pigments. A few weeks after injection, only the latter components remain in the skin ²¹. Pigments are insoluble species and they are absorbed by dermal macrophages ²². These cells store them until they die. Then, the pigments are temporary free but are phagocytized again by a new macrophage ²². This cycle of “death – release – recapture” explains the stability of the tattoos over the years. Fibroblasts have also been proposed to play a minor role in the maintenance of tattoos ²³.
Data on the impact of tattoos on health are mostly available for immediate cutaneous adverse
reactions. Reported effects include risks of infection and inflammation<sup>24-26</sup>, as well as diffusion
of the different ink's component soon after tattooing<sup>27, 28</sup>. Less information is available on the
adverse effects of the pigments. These compounds, often used in mixture, exhibit a large variety
of chemical and physical properties<sup>29</sup>. Black pigments mostly consist of carbon black<sup>30</sup>.
Titanium dioxide is used in white inks or to enhance the color strength of other pigments<sup>31, 32</sup>.
Colorful pigments can be inorganic derivatives. However, with the notable exception of iron
oxides, they are not used in the European Union since the concentration of several metals
(cadmium, chromium, cobalt, etc.) in tattoo inks has been limited to sub-ppm concentrations<sup>33</sup>
because of toxicological issues. Currently, colored tattoo pigments are thus mostly organic
species<sup>32</sup>. They are also strongly regulated in the European Union that published a list of
pigments that can be used, in a maximal concentration of 0.1 % in inks. In addition, two
pigments, phthalocyanines Pigment blue 15:3 and Pigment green 7, are now banned because
the toxicity. A wide variety of chemical structures have been designed to control the light
absorption properties and make various colors available, while being as resistant as possible to
fading. Several types of organic pigments are used, including diazo compounds,
phthalocyanines and polycyclic derivatives such as quinacridone or diketopyrrolopyrroles<sup>31</sup>.

Assessment of the toxicity of organic pigments in skin is an important task. It should include
the study of the impacts of their possible degradation products. It is indeed important to consider
that pigments located in the dermis are not inert but may undergo chemical and physical
transformations during the long period they remain in the skin. In the current project, we
hypothesized that small particles and soluble degradation products released upon aging of
pigments may be first produced in the dermis and then migrate into the epidermis, where they
could exhibit toxic effects. The presence of pigments in epidermal cells has actually been
documented for carbon black<sup>34</sup>. As a matter of fact, a number of putative adverse effects of
tattoos, such as inflammation, photosensitization or skin cancer, involve epidermal keratinocytes ³⁵⁻³⁷. The present work addresses this issue for three compounds: pigment orange 13 (PO13), pigment red 122 (PR122) and pigment red 254 (PR254). The motivation for this choice was that red tattoos are the most associated with cutaneous adverse effects ⁸, ¹⁰, ³⁸.

Secondly, as presented below, we found that these three pigments were the most toxic *in vitro* in a preliminary screening experiment. Two possible degradation pathways were studied *in vitro*. First, we extended to PR122 and PR254 the approach recently applied to the degradation of PO13 ³⁹ in aqueous suspension exposed to simulated sunlight used as a model of decomposition in sun-exposed skin areas ⁴⁰. Secondly, we asked the question of the decomposition of pigments in the phagolysosomes of macrophages. For this purpose, we suspended pigments in buffers mimicking the acidic and oxidizing conditions encountered in these organelles. In all experiments, the particles of aged pigments were characterized, putative degradation products were detected by high performance liquid chromatography associated to tandem mass spectrometry (HPLC-MS/MS), and several toxicological endpoints (cytotoxicity, oxidative stress, genotoxicity) were investigated in the human keratinocyte cell line HaCaT.

## MATERIALS AND METHODS

**Chemicals.** Pigment red 254 (PR254), Pigment red 122 (PR122), Pigment green 7 (PG7), Pigment blue 15:3 (PB15:3), Pigment yellow 74 (PY74) and Pigment violet 23 (PV23) were purchased from Kama Pigments (Montréal, Canada), and Pigment orange 13 (PO13) from Santa Cruz Biotechnology (Dallas, USA). The water used for the preparation of suspensions and of HPLC solvents was purified in a MilliQ direct 8 system (Millipore). Acetonitrile (Carlo Erba) used for HPLC mass spectrometry analyses was of LC-MS grade. Ammonium formate and formic acid (Aldrich) used for the HPLC buffers were of the highest purity available.

**Photoaging of pigments suspensions.** PR254 and PR122 were photoaged as previously performed for PO13 ³⁹. Suspensions of PR254 and PR122 were prepared in water at 10 mg mL⁻¹
and 0.1 mg mL⁻¹, respectively. Briefly, pigment particles were dispersed by sonication in a
cuphorn instrument (BioBlock Scientific, France). Pigment suspensions were transferred,
together with a 0.7 cm magnetic bar, into 1×1×3.5 cm spectrophotometer quartz cuvettes sealed
with a screw cap. The samples were placed, under constant magnetic stirring, in a QSUN XE-
1 Xenon Test Chamber (LX-5080TM, Qlab, USA) for 96 h at an intensity of 1.44 W m⁻² nm⁻¹
measured at 420 nm. The temperature within the sample was set at 40 °C. Two control samples
were also prepared (i) a pigment suspension incubated under agitation for 96 h at 40 °C in the
dark, and (ii) a pristine suspension kept in the dark at 4 °C. Aliquot fractions (500 µL) of the
suspensions were centrifuged (8000xg, 10 min) in filtering tubes bearing a 0.2 µm nylon
membrane (VWR). Another aliquot fraction of suspension (100 µL) was centrifuged (8000xg,
10 min) and the liquid fraction was discarded. Hundred µL of octanol were added. The sample
was vortexed and transferred to a 0.2 µm filtering tube. After centrifugation, the filtrate was
transferred to HPLC vials.

**Degradation of pigment in reconstructed macrophage phagolysosome medium.** A
solution simulating the macrophage phagolysosome environment was reproduced based on
previous works ⁴¹⁻⁴³. It was composed of 50 mM citric acid (VWR), 5 mM NaH₂PO₄, 5 mg mL⁻¹ bovine immunoglobulins (Ig), 10 mM H₂O₂ (Sigma-Aldrich) and 100 mM KOH (Fluka) at pH 5.5. First, a suspension with a 2X pigment concentration, i.e. 20 mg mL⁻¹ for PR254 and 0.2 mg mL⁻¹ for PR122, was prepared in a 2X pH 5.5 solution containing citric acid, KOH and NaH₂PO₄. Next, pigment particles in suspension (5 mL) were dispersed by sonication for 15 min and at an amplitude of 60% in a cuphorn instrument. The temperature was kept at 4°C by a thermostated water circulation. The final pigment suspension was obtained by adding immunoglobulins (Ig), H₂O₂ and water in such a way as to reach the expected salts and pigment concentrations (10 mg mL⁻¹ for PR254 and 0.1 mg mL⁻¹ for PR122). Two control samples were also prepared (i) a pigment suspension prepared only in water (control) and (ii) a pigment
suspension with Ig-free phagolysosomal medium (PGL w/o Ig). The control in plain water was a pigment suspension prepared in Milli-Q water at 2X concentration, then sonicated with the same parameters. The suspension was then diluted by half in Milli-Q water to obtain the same pigment concentration as the pigment suspension in phagolysosomal (PGL) medium. The different suspensions were transferred to glass bottles and incubated at 40°C in the dark, for 10 days, with continuous agitation provided by a magnetic bar and a magnetic stirrer. Halfway through the aging process, a 30% H₂O₂ solution was added to the mixture to replenish the quantity initially prepared. The same protocol without the incubation period was used to prepare the controls at t 0h. Aliquot fractions of each of the suspensions were also centrifuged (8000xg, 10 min) in 0.2 µm filtering tubes and transferred to HPLC vials.

**Physicochemical characterization.** Measurements of zeta potential and hydrodynamic diameter of pigment particles were performed on a Litesizer 500 particle analyzer (Anton Paar GmbH, Graz, Austria) using electrophoretic and dynamic light scattering, respectively. For the former technique, the photoaged pigment suspension, its two controls and the control of PGL aging sample were diluted in 1/1000 Dulbecco's Phosphate Buffered Saline (DPBS, Gibco, Illkirch, France). As they already contained salts, pigment suspensions in PGL and PGL w/o Ig samples were directly diluted in water. For DLS, samples were analyzed at different dilutions in order to perform reliable measurements.

**Transmission electron microscopy (TEM).** An aliquot fraction (1 mL) of a 10 mg mL⁻¹ aqueous pigment suspension was sonicated for 15 min using a sonicator equipped with a cup-horn probe. To observe pigment particles, samples were prepared according to a previously described protocol⁴⁴ using the negative-mica-carbon staining flotation technique. Briefly, samples were absorbed to a carbon film, on a mica sheet. They were then stained with a 1% solution of uranyl acetate (AcUr) UO₂(CH₃COO)₂·2H₂O in distilled water (pH 4.2-4.5). Next, samples were transferred to a copper grid. Images were acquired under low dose conditions
($<$10 e-/Å²) with defocus values between 1.2 and 2.5 µm using a Tecnai 12 LaB6 transmission electron microscope at an accelerating voltage of 120 kV and using the Gatan Orius 1000.

**High performance liquid chromatography associated with electrospray tandem mass spectrometry (HPLC-MS/MS).** The filtrate of the pigment samples (20 µL) was injected in an HPLC system (ExionLC, SCIEX) equipped with a reverse phase HPLC column (EC 100/2 Nucleodur C18 HTec, 100 × 2 mm ID, 1.8 µm particle size, Macherey-Nagel, Düren, Germany). The temperature of the column oven was set at 50 °C. The HPLC elution was performed at a flow rate of 0.35 mL min⁻¹ in the gradient mode. The mobile phases were an aqueous solution of 2 mM ammonium formate containing 0.05% formic acid (A) and acetonitrile (ACN) with 0.05% formic acid (B). The initial composition of the gradient was pure A. The proportion of B was then increased to 10% in 12 min, set to 60% at 20 min and reached 100% at 22 min. The latter composition was maintained for 3 min. The outlet of the column was directed first to a UV diode array detector and then to the inlet of a 6500+QTrap mass spectrometer (SCIEX) operated in the positive electrospray ionization. Single stage mass spectrometry analyses (MS1) were performed with the second analysis quadrupole used as linear ion trap. The mass range was 50 to 1000. Additional experiments were performed in the product ion scan mode (MS2) aimed to record fragmentation mass spectra of compounds detected in MS1.

**Production, purification and detection of PR122 photoproducts.** A PR122 suspension in isopropanol (35 mL at 1 mg mL⁻¹) was aged with a XLS1600R3 solar simulator (Solar Light Company, PA) emitting the UV portion of sunlight for irradiation times of up to 241 hours. Filtered aliquot fractions (20 µl) of the liquid phase of the suspension were analyzed by HPLC with MS1 detection. Six photoproducts of interest (PPD1 to PPD6) were identified. At the end of the exposure, the irradiated suspension was collected, centrifuged and the supernatant was filtered at 0.2 µm with a syringe filter. The solvent was then evaporated to dryness using a rotary evaporator. The dried residue was solubilized in 3 mL ACN and filtered (0.2 µm) before
being injected in 600 µL fractions into a semi-preparative HPLC system equipped with a reverse-phase column (C18 nucleosil, 250 × 4 mm ID, particle size: 5 µm, Macherey-Nagel, Düren, Germany). A gradient similar to that applied to HPLC-MS/MS analyses was used, with the exception of the flow rate set at 1 mL min⁻¹. Elution was monitored by a UV detector set at 230 nm. Five fractions were collected at different retention times (24.8, 26.8, 27.1, 27.5 and 29.9 min). HPLC-MS/MS analysis confirmed that the fractions corresponded to PPD1, PPD3, PPD4, PPD5 and PPD6. After being purified by a second purification under the same conditions, the photoproducts were solubilized in dimethyl sulfoxide (DMSO). An aliquot fraction of the PPD solutions (20 µL) was injected in the HPLC-MS/MS system with diode array detector set between 250 and 300 nm. The concentration of each photoproduct was inferred from the area of the corresponding peak compared to a calibration curve obtained by injecting increasing volumes (20 to 80 µL) of a 50 µM solution of acridanon (Aldrich).

In another experiment, 30 mL of aqueous suspension of PR122 was prepared in water at 0.1 mg mL⁻¹ and transferred in a quartz tube. The suspension was then photoaged in the QSUN XE-1 Xenon Test Chamber under the same condition as those used for photoaging the pigment suspensions in cuvettes. A fraction of the photoaged suspension (10 mL) was filtered (0.2 µm) and lyophilized. The dried product was suspended in water (3 mL). Another aliquot fraction (10 mL) was directly lyophilized and the resulting residue was suspended in isopropanol (3 mL). Half of this sample was concentrated to 200 µl and filtered. Both the filtered (0.2 µm) aqueous and isopropanol extracts were analyzed by HPLC-MS/MS. These experiments were performed in the product ion scan mode (MS2) in order to detect products exhibiting molecular weights of 225, 228, 278, 374 and 392 g mol⁻¹. A filtrate of pristine PR122 suspension was also analyzed under the same conditions as the photoaged samples.

**Cell culture.** HaCaT cells (CLS, Eppelheim, Germany), an immortalized keratinocytic cell line, were cultured in high glucose Dulbecco's modified Eagle's medium with Glutamine
(DMEM + GlutaMAXTM; 31966021, Gibco, Illkirch, France) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 UI mL⁻¹ penicillin and 0.1 mg mL⁻¹ streptomycin (Sigma), and maintained under an atmosphere of 5% CO₂ at 37 °C.

**Cell viability assay.** On the day prior to the treatment, HaCaT cells were seeded into 96-well plates (2.5×10⁴ cells/well) in culture medium. To study the cytotoxicity of several organic pigments, 100 µL of suspensions containing different concentrations of PG7, PB15:3, PV23, PY74, PO13, PR254 and PR122 (3×10⁻³ to 3 µg mL⁻¹) were added on the next day, for 24 h. To study the impact of the two types of aging on pigment cytotoxicity, 100 µL of suspensions containing different concentrations of pigment (3×10⁻³ to 100 µg mL⁻¹ for PR254 and 3×10⁻⁵ to 1 µg mL⁻¹ for PR122) or of PR122 photoproducts (1 to16 µM) were added on the next day, for 24 h. The filtrates of photoaged pigment suspensions (<0.2 µm) were also tested at different concentrations by performing the same dilutions as those done for the pigment suspensions. At the end of the treatment, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) solution was added in order to reach a concentration of 0.45 mg mL⁻¹ in each well. After 2 h at 37 °C, medium was discarded and formazan crystals were dissolved in 100 µL DMSO. Absorbance was measured at 550 nm (SpectraMax M2, Molecular Devices). Possible interferences of pigments with the assay were taken into account by measuring the absorbance in exposure medium without adding MTT for each experimental condition. The obtained values were subtracted from those obtained in the presence of the probe.

**Detection of reactive oxygen species by CM-H₂DCFDA.** Production of intracellular ROS content was monitored using CM-H₂DCFDA (5(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester, Invitrogen, France) assay. On the day prior to the treatment, HaCaT cells were seeded into black 96-well plates (3.5 × 10⁴ cells/well) in culture medium. On the next day, cells were rinsed with DPBS and incubated for 1h with a DPBS solution containing 5 µM CM-H₂DCFDA and 0.2% DMSO. As control, wells were only
incubated with a DPBS solution containing 0.2% DMSO. At the end of the incubation, cells
were rinsed with DPBS. High glucose DMEM without phenol red (Gibco, Illkirch, France)
supplemented with 4 mM GlutaMax (Gibco), 1 mM sodium pyruvate (Sigma), 10% FBS, 100
UI mL⁻¹ penicillin and 0.1 mg mL⁻¹ streptomycin was added in wells, followed by a 45-minute
incubation. Then, medium was discarded and suspensions containing different concentrations
of pigment (3×10⁻³ and 3×10⁻² µg mL⁻¹ for PR254 and 1 µg mL⁻¹ for PR122) were added. The
filtrates of pigment suspensions (<0.2 µm) were also tested at different concentrations by
performing the same dilutions as those done for the pigment suspensions. As positive control,
a solution with 250 µM *tert*-butyl hydroperoxide (tBuOOH, Luperox, Sigma) was added to
wells. After a 4-hour treatment, the fluorescence of fluorescein was measured at λexc/λem 488
nm/535 nm (SpectraMax M2, Molecular Device). Potential interferences of pigment particles
were taken into account by measuring the fluorescence of cells exposed to pigment and
previously incubated with a solution containing 0.2% DMSO without CM-H₂DCFDA. The
obtained values were subtracted from those obtained in the presence of the probe. Results were
normalized by dividing the difference between the measured fluorescence intensity for a
specific condition and the mean of that for the vehicle control by the difference between the
mean for the positive control and the mean for the vehicle control. Values were expressed in
percentage.

**Detection of DNA strand breaks by Comet assay.** On the day prior to the treatment, HaCaT cells were seeded into 12-well plates (3×10⁵ cells/well) in culture medium. On the next day, suspensions containing PR122 or PR254 at 0.03 µg mL⁻¹ were added. At the same time, a solution containing the filtrate obtained from PR122 or PR254 suspension at 0.03 µg mL⁻¹ was also added for 24h. It was obtained by performing the same dilutions as those done for the tested pigment suspensions. Next, the plates were incubated for 24 h. As positive control for the assay, methyl methanesulfonate (MMS) at 30 µg mL⁻¹, was used to induce DNA breaks. At the end
of the exposure, medium of wells was discarded. The wells were rinsed with DPBS. Cells were
recovered with trypsin-EDTA and stored at -80 °C in a storage buffer. The comet assay was
performed in its alkaline version. Briefly, microscope slides were coated with 1% normal
melting point agarose (NMA) and allowed to dry. Fifty µL of cell suspensions were mixed with
450 µL of prewarmed 1% low melting point agarose (LMPA) and then, 70 µL of these mixtures
were deposited over the agarose layer, and the LMPA/cells mix was allowed to solidify on ice.
After solidification of the gels, the slides were immersed for 1 h in cold lysis solution (2.5 M
NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100 with pH 10) in the dark. Slides were
rinsed three times with DPBS for 5 min. DNA was then allowed to unwind for 30 min in cold
alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA). Electrophoresis was
performed in a field of 1.2 V cm⁻¹ and 300 mA current for 30 min in the same solution. Slides
were rinsed twice with DPBS for 10 min. Samples were stained with GelRed (Biotium, San
Francisco, USA) for scoring. At last, 50 comets per slide were analyzed under a fluorescence
microscope (Zeiss) equipped with a LED emitting light with 380-770 nm wavelength (XCite
Xylis, Model No. XT720L, Excelitas Canada Inc., Kitchener, Canada) at ×20 magnification.
Percentage of tail DNA was measured by using Comet IV software (Perceptive Instruments,
Suffolk, UK).

**Statistics.** GraphPad Prism 9 software (Boston, USA) was used for the statistical analyses of data obtained for cell viability, ROS production, DNA strand breaks generation and particle hydrodynamic diameter measurement. Kruskal-Wallis tests followed by Dunn's multiple comparisons were used to analyze data from dose-course studies. Kolmogorov-Smirnov tests were applied to the comparison of results arising from different types of samples. Results were considered significant with *p* ≤ 0.05. For the statistical analysis of the toxicity of PR122 photoproducts, viability values were log transform based on the assumption of an exponential dose-effect. The non parametric Spearman correlation coefficient between these log-
transformed data and the photoproducts concentration was calculated and *p* values were
determined.

RESULTS

In the present work, we were interested in the links between the degradation of tattoo
pigments and their toxicological properties. In a preliminary screening step, we selected three
toxic compounds, and then studied their degradation in aqueous suspension upon photoaging
or incubation in phagolysosomal-like medium. The impact of the resulting alterations in terms
of toxicity was studied in the whole suspensions or in their filtrates.

## Properties of pristine pigments

Cytotoxicity

In a first step, we compared the toxicity of seven different pigments of different color and
exhibiting various chemical structures. The products were PG7, PB15:3, PV23, PY74, PR254,
PR122 and PO13. Their cytotoxicity was evaluated over a large range of concentrations in the
human keratinocyte cell line HaCaT by using the MTT assay. The results showed that the three
most toxic pigments were PR122, PR254 and PO13 (Figure 1). For this reason, the work then
focused on these three compounds.
<figure><img src="image_3.png" /><figcaption>**Figure 1:** Cytotoxicity of series of pigments suspended in water in HaCaT cells. Cells were exposed to the pigments for 24h. Cell viability was assessed by the MTT assay. Results were expressed as the mean of normalized cell viability ± standard deviation (n=3).</figcaption></figure>

## Physical characterization

PR254, PR122 and PO13 pristine particles were characterized by transmission electronic microscopy (Figure 2). The three pigments were found to be composed of submicrometric particles. They were detected as agglomerates of primary particles exhibiting a length of approximately 200, 250 and 350 nm for PR122, PR254 and for PO13, respectively. The particles of PR122, and to a lesser extent those of PO13, appeared rod-shaped. PO13 particles were composed of fused 100 nm sub-particles. PR254 particles were less regularly shaped.
<figure><img src="image_4.png" /><figcaption>**Figure 2:** Transmission electron microscopy analysis of PR254, PR122 and PO13 particles. The white bars on the left lower corners of the images correspond to 200, 100 and 50 nm at magnification of 2900, 11000 and 23000, respectively.</figcaption></figure>

## Effect of photoaging

PR254 and PR122 were subjected to artificial photoaging in a test chamber at 40°C under the light emitted by a filtered Xenon lamp. The spectrum and the intensity mimicked a tropical exposure. Experiments lasted 96h. Temperature controls were prepared by incubating suspensions in the dark at 40°C for the same time. In addition to the complete suspension, physical and toxicological characterizations were also performed on fractions filtered at 0.2 µm to collect information on the small particles and the soluble degradation products.

## Impact of photoaging on PR254

### Physical and chemical properties

The aged suspension was first characterized by DLS. The distribution in hydrodynamic diameter was only slightly different for the photoaged and the temperature control suspensions compared to the pristine sample. The median values were found to be 307, 307 and 281 nm, respectively (Figure 3a). The effect of photoaging on the particle size in the 0.2 µm filtrates
was opposite to that observed in complete suspensions (Figure 3b). The median diameter for the pristine and control suspensions was 98 nm in both cases while it decreased to 91 nm in photoaged samples. The polydispersity was also lower in the photoaged suspension. The impact of photoaging of PR254 was also studied in terms of surface charge of the particles. The zeta potentials were similar, -45.6 mV on the average, in pristine, control and photoaged samples (Supporting information Table S1).

<figure><img src="image_5.png" /><figcaption>**Figure 3:** Size distribution determined by DLS in suspensions of pristine PR254, in photoaged and control suspensions: a) non filtered suspensions, and b) obtained by filtration of suspension at 0.2 µm.</figcaption></figure>

A last characterization of the filtrates of photoaged suspensions was the search of soluble photoproducts by HPLC associated to a mass spectrometer recording single stage spectrum (MS1). The chromatograms showed tiny peaks present in photoaged PR254 samples and not in the controls. However, they were observed mostly at short exposure times. A few products appeared to be produced in a time-dependent way, but still in low amounts. In addition, they were not observed in all experiments. We also performed an octanol extraction of the particles but could not detect specific photoproducts. In addition, we could neither identify nor isolate
typical or abundant photoproducts upon aging of large amounts of PR254 suspended in isopropanol. It can be concluded that photoaging of PR254 did not induce an efficient formation of photoproducts.

## Toxicological impact

The impact of photoaging on the toxicity of PR254 was first investigated in terms of decrease in viability, measured by the MTT assay. As observed for the pristine suspension, the loss of viability induced by the temperature control was observed for concentration larger than 0.003 µg mL⁻¹ (Figure 4a). A very similar trend was observed for the photoaged samples. The difference in median values of viability between control and photoaged samples was statistically significant at none of the tested concentrations. They ranged between 75% viability and 45% at 0.003 and 100 µg mL⁻¹ for the control suspensions, and 77 and 51% at the same concentrations for the photoaged suspensions. The same similarity of viability between cell samples exposed to the control or the photoaged suspensions was obtained for the filtrates (Figure 4b). A significant loss of viability was observed above 0.3 µg mL⁻¹ but no difference was observed between the temperature control and the photoaged samples.
<figure><img src="image_6.png" /><figcaption>**Figure 4:** Cytotoxicity of control and photoaged suspensions of PR254. a) cells were exposed to increasing concentrations of suspension and b) cells were exposed to increasing amounts of filtrate prepared from the suspensions. After 24h, cell viability was assessed by the MTT assay. The results represent normalized cell viability and are expressed as the median ± 2^nd^ and 3^rd^ quartiles, with maximum and minimum (n=9). $ indicates significant differences from control (Kruskal-Wallis test followed by Dunn's multiple comparisons, $ p ≤ 0.05$, $$ p ≤ 0.01$, $$$ p ≤ 0.001$, $$$$ p ≤ 0.0001). Kolmogorov-Smirnov test was also performed and no difference between photoaged PR254 and temperature control at the same concentration of pigment was found.</figcaption></figure>
We then investigated two other important toxicological endpoints. The first was the induction of oxidative stress in cells exposed to suspensions of PR254. Neither the control nor the photoaged samples were found to favor the release of reactive oxygen species in the CM-H₂DCFDA assay (Supporting Information Figure S1). The next studied endpoint was the formation of DNA strand breaks assessed by the Comet assay. Again, no impact of the PR254 suspensions was observed (Table 1).

**Table 1:** Assessment of DNA strand breaks in HaCaT cells exposed to control or photoaged suspensions of PR254 and PR122. Cells were exposed to different pigment suspension (µg mL⁻¹) for 24 hours. DNA strand breaks was assessed by using the Comet assay. Methyl methane sulfonate (MMS) was used as a positive control. Results are expressed by tail intensities in % as the median ± 2nd and 3rd quartiles (n=9). No difference between the samples was found to be statistically significant, except between MMS and the solvent control (*** p ≤ 0.001).

<table><thead><tr><th rowspan="2">Pigment</th><th rowspan="2">Solvent</th><th colspan="2">Suspension</th><th colspan="2">Filtrate</th><th rowspan="2">MMS</th></tr><tr><th>Control 0.03</th><th>Photoaged 0.03</th><th>Control 0.03</th><th>Photoaged 0.03</th></tr></thead><tbody><tr><td>PR254</td><td rowspan="2">3.4±1.3</td><td>5.4±2.0</td><td>4.9±1.1</td><td>4.2±1.7</td><td>4.4±1.7</td><td rowspan="2">57.2±6.0***</td></tr><tr><td>PR122</td><td>3.5±1.4</td><td>3.6±1.2</td><td>3.3±1.1</td><td>3.4±0.7</td></tr></tbody></table>

## Impact of photoaging on PR122

### Physical properties

The size of the particles of PR122 determined by DLS (Supporting Information Figure S2) increased from 174.1 to 222.0 nm when the suspensions were left for 96h at 40°C (temperature controls). This difference was no longer significant for photoaged samples where the median size was 204.7 nm.

### Formation or photoproducts

In contrast to PR254, PR122 was found to yield several photoproducts upon photoaging. In a first experiment, we exposed a suspension of the pigment in isopropanol to the UV light
emitted by a solar simulator for 10 days. Analysis of the raw photodegradation mixture showed the presence of 6 new peaks on the HPLC-MS1 chromatogram (Figure 5), which were absent at t0. In spite of its poor shape, the peak with a retention time (Rt) of 14.1 min consisted of only one photoproduct (PPD1). PPD2 (Rt 15.4 min) and PPD5 (Rt 18.6 min) appeared as well-defined peaks. Two compounds (PPD3 and PPD4) were found to co-elute as a large peak at 16.5 min. They were separated in a subsequent semi-preparative HPLC purification step. The two peaks that eluted last, with respective Rt of 18.9 and 19.3 min, were probably in equilibrium one with the other. It was not possible to isolate one without an equal amount of the other. Both products were thus referred to as PPD6. In order to gather structural information, the sample photoaged in isopropanol was then injected in HPLC with the mass spectrometer set in the fragmentation mass spectrometry mode (MS2). UV absorption spectra could also be recorded on-line by a diode array detector. Tentative chemical structures could be proposed based on the MS1, MS2, and UV-visible absorption (Supporting information Figure S3 to S8). All the photoproducts, except PPD2, could be isolated and purified. Unfortunately, the collected amounts were too low to apply other characterization techniques such as NMR or infrared spectroscopies.

<figure><img src="image_7.png" /><figcaption>**Figure 5:** Chromatograms obtained upon analysis of a photoaged suspensions of PR122. Exposure was performed in isopropanol before (-) and after 241 h (-) exposure to UV radiation. The inset shows tentative chemical structures for the corresponding products.</figcaption></figure>
These chromatographic and mass spectrometry features were used to search for the
photoproducts in an aqueous suspension of PR122 photoaged in the test chamber. A larger
volume of suspension than that typically used in photoaging experiments was used but all other
experimental conditions were the same. The water of the suspension was removed by freeze-
drying and the obtained residue extracted by isopropanol or water. HPLC-MS/MS with
detection in the MS2 mode unambiguously showed the presence of PPD2 and PPD5 in the
organic extract (Supporting information Figure S9). Traces of the two peaks corresponding to
PPD6 were also detected when specific fragments were searched in the data in order to increase
the sensitivity (Supporting information Figure S10). PPD2 was also detected in the aqueous
extract but in an almost 10-fold larger concentration than in the isopropanol extract (Supporting
information Figure S11).

Toxicological impact of photoaging

Photoaging modulated neither the cytotoxic impact of PR122 suspension nor that of the
corresponding filtrates (Supporting Information Figure S12) in HaCaT cells. Exposure to
PR122 suspensions led to a statistically lower viability compared to the solvent control for
concentrations above 0.03 µg mL⁻¹ whether they were photoaged or not. One exception was at
0.0015 µg mL⁻¹ but most likely because of an outlier low value for the photoaged sample. The
filtrates led also to a similar loss of viability whether they arose from a control or a photoaged
suspension. We then investigated the ability of control and photoaged suspensions at inducing
DNA damage. No difference in the frequency of strand breaks was observed between cells
exposed to the solvent control and the control or photoaged suspensions (Table 1). The only
adverse effect induced by photoaging was a larger capacity of the PR122 suspension at inducing
oxidative stress at 1 µg mL⁻¹, as shown by the CM-H₂DCFDA assay (Figure 6). It should be
noticed the treatment time (4h) of cells in this assay is shorter than in other tests because the
fluorescence signal reaches a plateau between 6 and 24h of incubation with the probe.
<figure><img src="image_8.png" /><figcaption>**Figure 6:** Induction of ROS in HaCaT cells exposed to control and photoaged PR122 suspensions. Cells were exposed to PR122 for 4 hours. ROS production was assessed by using the CM-H₂DCFDA assay. The results represent the normalized fluorescence intensities and are expressed as the median ± 2nd and 3rd quartiles, with maximum and minimum (n=9). tBuOOH was used as a positive control. $\$ indicates significant differences from vehicle control (Kruskal-Wallis test followed by Dunn's multiple comparisons, $ p \le 0.05$, $$$ p \le 0.0001). * indicates significant differences between photoaged PR122 and temperature control at the same concentration of pigment (Kolmogorov-Smirnov test, ** $p \le 0.01$).</figcaption></figure>

In a last set of experiments, we investigated the cytotoxicity of the photoproducts isolated from the suspension of PR122 in isopropanol exposed to UV radiation for 10 days. This study did not involve PPD2 that was present in too small amounts and could not be isolated as a pure product. Prior to this work, we determined the concentration of the different solutions of photoproducts by HPLC with UV detection. For this purpose, we drew a calibration curve, using acridanon as a reference compound because its structure was present in most PR122 photoproducts. HaCaT cells were then exposed to increasing concentrations of photoproducts in solution in DMSO. A first experiment was made at 1 µM and a second at 6 µM, except PPD6 for which only low amounts were available and could be tested only at 2 µM. The last tests
were performed at the largest possible concentrations based on the amount of purified
photoproduct available. The statistical significance of the effect was assessed with the
assumption of an exponential decrease in viability with increasing concentration. With the
exception of PPD5, all photoproducts were found to induce a significant loss of viability.
(Figure 7). The decreasing of order of cytotoxicity (and the *p* values) was the following: PPD6
(*p*<0.001) > PPD4 (*p*<0.001) > PPD1 (*p*<0.001) > PPD4 (*p*<0.01).

<figure><img src="image_9.png" /></figure>

**Figure 7:** Cytotoxicity of PR122 photoproducts in HaCaT cells. Cells were exposed to PR122 photoproducts for 24h. Loss of viability was assessed by the MTT assay. Results are expressed as the mean of normalized cell viability ± standard deviation (n=3). Calculation of the non parametric Spearman correlation coefficient from the log-transformed values showed the statistical significance of the loss of viability with the exception of PPD5.

**Simulation of phagolysosomal degradation**

In the last part of the work, we investigated the degradation of the three pigments under
conditions mimicking those encountered in the phagolysosomes of the macrophages where
pigment particles are trapped over a long period. The medium of phagolysosomes (PGL) is
characterized by acidic and oxidizing conditions, and the presence of proteins involved in the
phagocytosis of antigens <sup>45</sup>. Consequently, the solutions used for the aging experiments
consisted of a citrate and phosphate buffer set at pH 5.5 and spiked with hydrogen peroxide and
immunoglobulins (Ig). Two control conditions were used: a temperature control made of a
suspension in water and a sample similar to PGL but without Ig. Samples were collected immediately after preparation of the suspension or after 10 days of incubation at 40°C. Hydrogen peroxide was added after 5 days to compensate for degradation.

## Physical and chemical characterization

DLS analyses (Table 2) showed that after 10 days, namely 6 days more than in the photoaging experiments, the hydrodynamic diameter had significantly increased compared to t0 in the case of PR254 (p≤0.001) and PO13 (p≤0.001) suspended in plain water. No difference was observed for PR122. For the three pigments, a significant larger value (p≤0.001) was observed at t0 for the PGL than the temperature control samples. The same was true at D10 for PR254 and PR122 but not PO13. For PGL samples, an increase in size was observed between t0 and D10 for PR122 (p≤0.001) and PO13 (p≤0.001) but not PR254. The filtrates of the suspensions were analyzed by HPLC-MS1 but no significant and reproducible difference could be observed between the chromatograms of t0 and D10 samples, ruling out the presence of degradation products.

<table><caption>**Table 2:** Hydrodynamic diameters measured in suspensions of PR254, PR122 and PO13 under conditions mimicking the phagolysosomal medium (PGL) and in temperature control samples and samples without immunoglobulins (PGL w/o Ig). The values are median ± 2nd and 3rd interquartiles range (n=9). Statistical significance of the differences was calculated between aged PR122 and control at the same incubation time (Kolmogorov-Smirnov test, *** p ≤ 0.001).</caption><thead><tr><th>Pigment</th><th>Control t0</th><th>Control D10</th><th>PGL t0</th><th>PGL D10</th><th>PGL w/o Ig t0</th><th>PGL w/o Ig D10</th></tr></thead><tbody><tr><td>PR254</td><td>148±56</td><td>307±72</td><td>499±127***</td><td>460±150***</td><td>499±162***</td><td>499±162***</td></tr><tr><td>PR122</td><td>126±69</td><td>116±75</td><td>241±78***</td><td>499±117***</td><td>n.d.</td><td>n.d.</td></tr><tr><td>PO13</td><td>307±115</td><td>499±204</td><td>391±166***</td><td>460±150</td><td>424±138***</td><td>541±184</td></tr></tbody></table>

Concerning the impact of immunoglobulins on the particle size, no difference was observed for PR254 between the PGL samples, whether at t0 or D10, and the PGL w/o Ig suspension (Table 2). For PO13, a small but significant increase (p≤0.01) was observed between t0 and
D10 in the absence of Ig. No result could be obtained for the suspension of PR122 in the PGL medium without immunoglobulin. Under this condition, a rapid aggregation of the particles was observed and the resulting agglomerates migrated to the surface of the suspension (Supporting information Figure S13). This was not the case for suspensions in PGL medium and aggregation was much less pronounced in water. Other noticeable results were obtained for the Zeta potential. Incubation of PR254, PR122 and PO13 suspensions in complete PGL medium led to a sharp decrease (in absolute value) to almost 0 mV at both t0 and D10 (Figure 8). This effect was no longer observed when the control suspensions were compared to suspensions prepared in PGL medium lacking Ig.

<figure><img src="image_10.png" /><figcaption>**Figure 8:** Zeta potential measured in suspensions of pigments in water, PGL medium and PGL medium without immunoglobulins (PGL w/o Ig). Results, expressed in mV, are the mean of three measurements.</figcaption></figure>

## Cytotoxicity

We then investigated the effect of 10 days of incubation in PGL on the cytotoxicity of PR254, PR122 and PO13. The tested concentrations were different from one pigment to the other to take into account the difference in cytotoxicity between the pristine compounds (PO13 < PR254 < PR122). A concentration-dependent loss of viability with the control samples was observed for PR122 (viability from 74 to 61% between 0.03 and 1 µg ml⁻¹) and PO13 (viability from 72
to 56% between 0.3 and 30 µg mL⁻¹) but not PR254 because of a large dispersion of the results (Figure 9). Similar trends were observed for the suspensions in PGL. A major difference was that, for the three pigments at all tested concentrations, the suspensions in PGL induced a significantly lower loss of viability than the control samples. For example, viability was 65% for PR254 control suspension at 3 µg mL⁻¹ and 96% for suspension in PGL. Corresponding values for the largest tested concentrations were 61 an 82 % for PR122 (1 µg mL⁻¹), and 56 and 79% for PO13 (30 µg mL⁻¹). Concerning the filtrates of these samples, we observed a trend toward a lower cytotoxicity of the filtrates of the suspensions in PGL compared to that from the control. Yet, this difference was statistically significant only for PR122 at 1 µg mL⁻¹ (Supporting information Table S2). Interestingly, toxicity of the suspension was restored to the values of the control when incubation of the pigments was performed in PGL in the absence of immunoglobulins (Table 3).

<figure><img src="image_11.png" /></figure>

**Figure 9:** Impact of phagolysosome-like environment on cytotoxicity of PR254, PR122 and PO13 in HaCaT cells. Cells were exposed to different pigment suspensions (µg mL⁻¹) for 24h. Viability was assessed by the MTT assay. The results are expressed as the median of normalized cell viability ± 2nd and 3rd quartiles, with maximum and minimum. * Indicates significant differences between aged pigment by phagolysosomal medium and control at the same concentration of pigment (Kolmogorov-Smirnov test, \*p ≤ 0.05, \*\* p ≤ 0.01, \*\*\*p ≤ 0.001, \*\*\*\*p ≤ 0.0001).
**Table 3:** Loss of viability of HaCaT cells exposed to increasing concentrations of pigment suspensions left for 10 days in water (control) or in PGL mixture without Ig (PGL w/o Ig). Cells were exposed to different pigment suspension (µg mL⁻¹) for 24h. Viability was assessed by the MTT assay. Values are expressed as mean cell viability ± standard deviation (n=6). No statistically significant difference was observed for samples incubated in water or in PGL w/o Ig with a specific pigment at a specific concentration.

<table><thead><tr><th colspan="6">PR254</th></tr></thead><tbody><tr><td>Control 0.03</td><td>PGL w/o Ig 0.03</td><td>Control 0.3</td><td>PGL w/o Ig 0.3</td><td>Control 3</td><td>PGL w/o Ig 3</td></tr><tr><td>63.4±9.8</td><td>66.0±1.7</td><td>47.2±0.4</td><td>46.9±0.9</td><td>55.4±5.9</td><td>51.4±3.5</td></tr></tbody><thead><tr><th colspan="6">PR122</th></tr></thead><tbody><tr><td>Control 0.03</td><td>PGL w/o Ig 0.03</td><td>Control 0.3</td><td>PGL w/o Ig 0.3</td><td>Control 1</td><td>PGL w/o Ig 1</td></tr><tr><td>75.4±3.2</td><td>96.8±5.6</td><td>59.6±3.0</td><td>91.7±2.2</td><td>56.6±0.9</td><td>84.4±2.8</td></tr></tbody><thead><tr><th colspan="6">PO13</th></tr></thead><tbody><tr><td>Control 0.3</td><td>PGL w/o Ig 0.3</td><td>Control 3</td><td>PGL w/o Ig 3</td><td>Control 30</td><td>PGL w/o Ig 30</td></tr><tr><td>69.9±5.9</td><td>74.9±7.9</td><td>52.6±6.5</td><td>47.0±9.0</td><td>53.8±11.2</td><td>46.1±5.1</td></tr></tbody></table>

DISCUSSION

The present study aimed to characterize adverse effects of tattoo pigments in keratinocytes mediated by degradation products, such as soluble derivatives or small-size particles. These species can easily migrate to the epidermis and induce deleterious responses in the cells, in particular in keratinocytes that represent the most abundant cell type. Two possible degradation pathways were investigated: i) the photodecomposition taking place in sun-exposed areas of the skin and ii) the modification of pigments in the phagolysosome where they are located inside macrophages. The work was performed with Pigment red 254 (PR254), Pigment red 122 (PR122) and Pigment orange 13 (PO13), selected as the most toxic in their pristine form among 7 products of different color and chemical nature. It should be noted that allergic reactions have
been reported to be the most frequent in red-tinted areas of tattoos<sup>8,38</sup>, which makes the study
of the toxicological properties of the corresponding pigments even more interesting.

The topic of photodegradation of organic pigments is highly relevant to tattoo toxicity<sup>40</sup> as evidence has been reported for the release of photoproducts in tattooed animals<sup>46</sup>. Unfortunately, only limited information on the nature of photodegradation products of tattoo pigments is available<sup>40, 47, 48</sup>. Results have been reported for the photolysis of Pigment yellow 74<sup>49</sup>, Pigment red 22<sup>50</sup> and acid red 87<sup>48</sup>, but these experiments were performed only under organic environment. Other studies involved the degradation of PB15:3 and PG7 by laser photolysis<sup>51, 52</sup>. In a previous work on PO13<sup>39</sup>, we developed a more relevant strategy for the study of photodecomposition of pigments. Pigments suspensions were prepared in pure water and the photoaging process mimicked equatorial conditions, with the temperature of the test chamber maintained at 40°C, and a spectrum and intensity of the light source similar to those of a zenithal exposure in both the UV and visible range. Information collected on this basis was thus more environmentally and physiologically relevant than in works involving pure UV and organic solution of pigments. Additional results were obtained in suspension in isopropanol but their relevance to photodegradation in aqueous mixture was verified. Photoaging has little effect on PR254 and PR122 particles in terms on size distribution and Zeta potential. These results contrast with those obtained under similar conditions with PO13. Formation of a nanometric fraction was observed in photoaged suspensions of PO13, with the average hydrodynamic diameter decreasing from more than 500 nm in the pristine state to 170 nm after photoaging<sup>39</sup>. The second investigated consequence of photoaging was the formation of soluble photodegradation products. This process can be relevant to cutaneous toxicity since some pigments have been shown to release toxic products such as aromatic amines<sup>47, 53</sup>. Our work of PO13 had led to the isolation and identification of a toxic photoproduct<sup>39</sup>. In contrast, PR254 did not generate specific photoproducts upon aging. This result is in line with the known
photostability of this pigment⁵⁴. An opposite trend was observed for PR122, a more photoreactive compound⁵⁴, that was found to give rise to the formation 6 photoproducts when irradiated in isopropanol. Three of them were also detected in more biologically relevant suspensions of PR122 photoaged in water in the test chamber. The yields of these photoproducts were yet approximately one order of magnitude lower than in the case of the PO13 photoproduct

³⁹.

These results shed some light on the different impacts of photoaging on the toxicological properties of pigments that were investigated either as complete suspension or after filtration at 0.2 µm. We did not observe a difference between the concentration-dependent loss of viability of HaCaT cells exposed to temperature control and photoaged suspensions of either PR254 or PR122. Similarly, we did not observe a difference between the filtrated fractions arising from control and photoaged suspensions of PR254 and PR122. Photoaged suspensions of the two red pigments and their filtrates induced neither DNA strand breaks nor ROS. One exception was a significant increase in the level of ROS in cells exposed to the largest investigated concentration of photoaged PR122 suspension. Again, PO13 was found to be more deleterious ³⁹ since photoaging increased the cytotoxicity of the filtrates of its suspensions. A first possible explanation was that PO13 was much more prone than PR254 and PR122 at releasing large amount of small size particles upon photoaging. Interestingly, a greater cytotoxicity has been shown for particles of same chemical composition but smaller diameter ⁵⁵⁻⁵⁷, partly because of an easier because small particles can be internalized by a wide variety of cellular mechanisms⁵⁸. The formation of photoproducts may also be involved in the modulation of the toxicological profile of pigment suspensions by photoaging. This was found to be a key finding for PO13 which was shown to degrade into a cytotoxic photoproduct capable of inducing oxidative stress and impacting metabolism of xenobiotics ³⁹. PR254 did not lead to the formation of photo-induced degradation product, which may also explain why the toxicity of its soluble fraction
was not affected by photoaging. The case of PR122 is more complex. Several photoproducts of this pigment were isolated and the impact of five of them on HaCaT cell viability was determined. Large differences in cytotoxic potential were observed but the most toxic one was among those detected in photoaged aqueous suspension. Yet, the formation of these toxic compounds did not impact the cytotoxic properties of photoaged PR122. A first explanation was that PR122 was the most toxic among the investigated pigments and that the additional effect of the photoproducts was not strong enough to have a detectable impact. Another explanation is that the yield of PR122 photoproduct was 10 times lower than that of PO13. Consequently, the concentration of PR122 photoproducts in photoaged suspensions was possibly too low to modulate the cytotoxicity of the pigment suspension. Further characterization of the PR122 photoproducts, currently hampered by the small amounts available, would be interesting, in particular in terms of oxidizing properties and activation of metabolism, as observed for the PO13 photoproduct ³⁹. Metabolism has actually been identified as a key process in the toxicity of tattoo pigments ⁴⁰.

Another possible degradation mechanism of pigments could be driven by the oxidative and acidic conditions encountered in the phagolysosomal environment of dermal macrophages ⁵⁹. The toxicological relevance of this process is strongly suggested by the cytotoxicity of metal-based pigments in cultured macrophages ⁶⁰, ⁶¹. We thus investigated whether a similar degradation pathway could occur with the three studied organic red pigments. In order to work under well-controlled conditions and with large amount of starting material, we chose to incubate pigments in suspension in a solution mimicking the medium of phagolysosomes. This cellular body results from the fusion of a lysosome with a phagosome and plays a major role in phagocytosis ⁶². The phagolysosomal environment is characterized by an acidic pH ⁶³, ⁶⁴ and the presence of oxidizing species ⁶⁵⁻⁶⁷. Therefore, we worked under conditions reported in the literature ⁴¹⁻⁴³ which involved a solution of citrate and phosphate set at pH 5.5, to which
hydrogen peroxide was added. The solution also contained immunoglobulins as a model of the
acid-stable proteins present in large amounts in lysosomal proteins⁶⁸.

The reconstructed phagolysosomal medium had a strong impact on the physical properties of the three studied pigments. The diameter of the PR254 and PO13 particles was larger in suspension in the PGL medium compared to plain water. This can be explained by the higher ionic strength in solutions containing potassium salts and citric acid compared to plain water. This results in an increase in polarity of the solvent, and consequently it favors hydrophobic interactions between organic pigment particles, which leads to larger hydrodynamic diameter. Removing Ig did not modify the size distribution, which shows that the increase in size in the PGL medium was mostly mediated by the electrolytes. The effect of the incubation in PGL was particularly strong for PR122. Suspensions of this pigment in PGL medium without Ig underwent a strong aggregation. The ability of quinacridones to form networks of stacked molecules thanks to intermolecular hydrogen bonds⁶⁹ could explain this greater stability of PR122 aggregates compared to PR254 and PO13. The addition of Ig prevented this process, but the average size of PR122 particles in the complete PGL medium was approximately 3 times larger than in water.

These observations of a strong agglomeration of pigment particles in PGL medium in the absence of Ig while the suspensions were more homogeneous and stable in their presence suggest that Ig were able to coat the primary particles by forming a protein corona<sup>70, 71</sup> and prevent further agglomeration. Formation of protein corona on tattoo pigment particles is not documented but information can be inferred from studies on organic nanoparticles<sup>71</sup>. First, it was shown that the binding of human serum albumin and fibrinogen resulted in loose “soft corona” on silica nanoparticles while a tightly bound “hard corona” was obtained with polystyrene nanoparticles<sup>72</sup>. More specifically, 100 nm polystyrene nanoparticles were found to efficiently bind Ig, especially when polystyrene was substituted with carboxylate group
providing negative surface charge ⁷³. This property may be relevant to the present pigment
particles that all exhibited negative zeta potential in their pristine state. These observations show
that Ig can bind to pigment particles and modify their surface properties. The hypothesis of the
formation of a protein corona was confirmed by the measurement of Zeta potential. For the
three pigments, this value ranged between -40 and -20 mV in water or in PGL medium without
Ig. When Ig were added, the Zeta potential became close to 0 mV whether measurements were
made at the beginning or the end of a 10-day incubation.

The aging in complete PGL medium did not increase the toxicological properties of pigments.
A very significant opposite trend was actually observed. At all investigated concentrations,
suspensions of pigments aged for 10 days in PGL medium were 20 to 30 % less cytotoxic than
controls i.e. pigment aged in suspension in water. When PGL medium where no Ig was added
was used, the cytotoxicity was similar to that observed for aqueous suspensions. This is in line
with the neutralization of the surface charge that we observed for pigment particles incubated
in PGL. The loss of toxicity of suspensions aged in PGL can thus be explained by the formation
of a protein corona on the particles, which could prevent direct interactions between the organic
molecules of the pigment and the cellular components, as suggested by the decreased toxicity
for organic ⁷⁴⁻⁷⁷ and inorganic ⁷⁸⁻⁸¹ nanoparticles. Ig were in particular found to decrease the
cytotoxicity of carbon nanotubes ⁸², which are hydrophobic particles like the presently studied
pigments. These results show that, in contrast to what is done in some studies ⁷⁸, ⁸³⁻⁸⁵, artificial
lysosomal fluid should contain proteins in their formulations, since it has long been known that
lysosomes are protein-rich organelles ⁶⁸, as protein-rich as the cytosol. With the well-known
ability of proteins to i) passivate the particles surfaces and ii) be able to bind to particle
degradation products such as metal ions or small organic compounds, this absence of proteins
may considerably bias the observations made in classical protein-free artificial lysosomal fluid.
These particle-binding properties of Ig should be taken into account in our observation of the
absence of soluble degradation product in the filtrates of the suspensions of the three
investigated pigments aged in PGL medium. Whether this is explained by a lack of reactivity
of the particles in reconstituted phagolysosomal medium or by a passivation of the surface by
Ig remains to be determined. Formation of a stable protein corona on pigment particles in
phagolysosomes may also have consequences in the only poorly investigated ⁸⁶, ⁸⁷ long-term
toxicity of tattoos. It may be proposed that free pigment particles released in the “death –
release – recapture” cycle ²² are passivated and exhibit a lower toxicity than the pristine pigmen
t.

CONCLUSION

The present work provided novel information on the fate of tattoo pigments. We extended our previous observations made on PO13 that exposure to sunlight could be at the origin of the degradation of pigment particle into nanoparticles and toxic soluble photoproducts, at least for PR122. Both types of degradation products could diffuse away from their original location in the dermis and reach internal organs through the blood flow or migrate to the adjacent epidermis. Such processes obviously depend on the molecular nature of the pigment. Among the three pigments we investigated, PO13 was found to be the most phototoxic, which points to a sensitivity of azoic derivatives. An additional process studied in this work was the phagolysosomal degradation. Using a reconstituted medium, we did not observe evidence of degradation of the pigments. In contrast, we obtained strong evidence for the formation of a protein corona by immunoglobulins which inhibited the adverse effects of the pigment. Whether this effect actually takes place in dermal macrophages remains to be shown but our results point to a major modulating role of corona. The observation of the release of photoproducts upon sun exposure and of a possible decreased toxicity as the results of formation of a corona of pigment particles deserve to be further explored in more complex skin models such as tattooed reconstituted skin ⁸⁸ or studies in tattooed animals ²³, ²⁷, ⁴⁶. In addition,
elucidation of the chemical structure of the photoproducts made possible the development of sensitive analytical tools for their quantification in skin and biological fluids of tattooed persons.

## ACKNOWLEDGMENTS

This work was supported by a grant from the French Agence Nationale de la Recherche (Tattooink project, grant # ANR-21-CE34-0025). The authors wish to thank Christine Saint-Pierre from the *Biomade* platform (IRIG/SyMMES, CEA-Grenoble, France) for her assistance in the DLS analyses. We also wish to thank Daphna Fenel for the electron microscopy analyses performed at the IBS Electron Microscope facilities at the (ISBG ; UAR 3518 CNRS CEA-UGA-EMBL) with support from the French Infrastructure for Integrated Structural Biology (FRISBI ; ANR-10-INSB-0005-02) and the GRAL Labex (CBH-EUR-GS (ANR-17-EURE-0003).

## ASSOCIATED CONTENT

**Data Availability Statement:** The data sets generated and analyzed during the current study are available from the corresponding author upon request.

**Supporting information:** Zeta potential of photoaged pigment particles; Viability of HaCaT cells exposed to filtrates of pigments aged in PGL; ROS level in cells exposed to photoaged PR254 suspensions; Impact of photoaging on the size of PR122 particles; Mass spectrometry characterization of photoproducts produced upon photoaging of a suspension of PR122 in isopropanol; Detection and characterization of PR122 photoproducts in extracts of a photoaged suspension of PR122 in water; Cytotoxicity of suspensions and filtrates of photoaged PR122; Stability of PR122 suspensions in PGL with or without immunoglobulins
REFERENCES

(1) Kluger, N.; Seité, S.; Taieb, C. The prevalence of tattooing and motivations in five major countries over the world. *Journal of the European Academy of Dermatology and Venereology* **2019**, *33* (12), e484-e486. DOI: 10.1111/jdv.15808.

(2) Schaeffer, K.; Dinesh, S. 32% of Americans have a tattoo, including 22% who have more than one. *Pew Research Center*. Pew Research Center, 2023. https://www.pewresearch.org/short-reads/2023/08/15/32-of-americans-have-a-tattoo-including-22-who-have-more-than-one/.

(3) Chiaramonte, F. *How Many People Have Tattoos? A Global Perspective on Who's Getting Inked*. Ink Studio AI, 2024. https://inkstudioai.com/blog/how-many-people-have-tattoos.

(4) Klugl, I.; Hiller, K. A.; Landthaler, M.; Baumler, W. Incidence of health problems associated with tattooed skin: a nation-wide survey in German-speaking countries. *Dermatology* **2010**, *221* (1), 43–50. DOI: 10.1159/000292627.

(5) Chalarca-Cañas, D.; Caviedes-Cleves, M.; Correa-Londoño, L.; Ospina-Gómez, J.; Velásquez-Lopera, M. Tattoos: risks and complications, clinical and histopathological approach. *An. Brasil. Dermatol.* **2024**, *99* (4), 491–502. DOI: 10.1016/j.abd.2023.07.004.

(6) Balaceanu-Gurau, B.; Apostol, E.; Caraivan, M.; Ion, A.; Tatar, R.; Mihai, M.; Popa, L.; Gurau, C.; Orzan, O. Cutaneous Adverse Reactions Associated with Tattoos and Permanent Makeup Pigments. *J. Clin. Med.* **2024**, *13* (2), 24. DOI: 10.3390/jcm13020503.

(7) Hutton Carlsen, K.; Serup, J. Photosensitivity and photodynamic events in black, red and blue tattoos are common: A 'Beach Study'. *Journal of the European Academy of Dermatology and Venereology: JEADV* **2014**, *28* (2), 231–237. DOI: 10.1111/jdv.12093.

(8) van der Bent, S. a. s.; Rauwerdink, D.; Oyen, E. m. m.; Maijer, K. i.; Rustemeyer, T.; Wolkerstorfer, A. Complications of tattoos and permanent makeup: overview and analysis of 308 cases. *Journal of Cosmetic Dermatology* **2021**, *20* (11), 3630–3641. DOI: 10.1111/jocd.14498.

(9) Wenzel, S. M.; Rittmann, I.; Landthaler, M.; Bäumler, W. Adverse Reactions after Tattooing: Review of the Literature and Comparison to Results of a Survey. *Dermatology* **2013**, *226* (2), 138–147. DOI: 10.1159/000346943.

(10) Gaudron, S.; Ferrier-Le Bouedec, M. C.; Franck, F.; D'Incan, M. Azo pigments and quinacridones induce delayed hypersensitivity in red tattoos. *Contact Dermatitis* **2015**, *72* (2), 97–105. DOI: 10.1111/cod.12317.

(11) Brusasco, M.; Spagnolini, S.; Mazzoni, L.; Magi, S.; Scarcella, G.; Stanganelli, I. Melanoma Arising in Tattoos: A Case Series and Scoping Review of the Literature. *Cancers* **2025**, *17* (5), 17. DOI: 10.3390/cancers17050767.

(12) Clemmensen, S.; Mengel-From, J.; Kaprio, J.; Frederiksen, H.; Hjelmborg, J. Tattoo ink exposure is associated with lymphoma and skin cancers - a Danish study of twins. *BMC Public Health* **2025**, *25* (1), 9. DOI: 10.1186/s12889-025-21413-3.

(13) Leijs, M.; Schaefer, H.; Rübben, A.; Cacchi, C.; Rustemeyer, T.; van der Bent, S. Cutaneous Malignancies in Tattoos, a Case Series of Six Patients. *Current Oncology* **2021**, *28* (6), 4721–4737. DOI: 10.3390/curroncol28060398.

(14) Lerche, C. M.; Heerfordt, I. M.; Serup, J.; Poulsen, T.; Wulf, H. C. Red tattoos, ultraviolet radiation and skin cancer in mice. *Exp Dermatol* **2017**, *26* (11), 1091–1096. DOI: 10.1111/exd.13383.

(15) Ricci, F.; Fania, L.; Antonelli, F.; Ricci, F.; Perino, F.; Di Lella, G.; Pallotta, S.; Fortes, C.; Panebianco, A.; Abeni, D. Melanoma on tattoos: a case study and review of the literature. *Eur. J. Dermatol.* **2022**, *32* (6), 703–708. DOI: 10.1684/ejd.2022.4353.

(16) Ricci, F.; Paradisi, A.; Maier, S. A.; Kovacs, M.; Podda, M.; Peris, K.; Abeni, D. Melanoma and tattoos: a case report and review of the literature. *Eur J Dermatol* **2018**, *28* (1), 50–55. DOI: 10.1684/ejd.2017.3184.
(17) Kluger, N.; Koljonen, V. Tattoos, inks, and cancer. *The Lancet Oncology* **2012**, *13* (4), e161-e168.
DOI: 10.1016/S1470-2045(11)70340-0.

(18) Liljedahl, E. R.; Engfeldt, M.; Nielsen, K.; Joud, A.; Nielsen, C. Tattoos and cutaneous squamous cell carcinoma: a population-based case-control study. *Eur J Epidemiol* **2025**, *40* (4), 451-461. DOI: 10.1007/s10654-025-01230-z.

(19) Gurnani, P.; Williams, N.; Al-Hetheli, G.; Chukwuma, O.; Roth, R.; Fajardo, F.; Nouri, K. Comparing the efficacy and safety of laser treatments in tattoo removal: A systematic review. *Journal of the American Academy of Dermatology* **2022**, *87* (1), 103-109. DOI: 10.1016/j.jaad.2020.07.117.

(20) Hernandez, L.; Mohsin, N.; Frech, F. S.; Dreyfuss, I.; Vander Does, A.; Nouri, K. Laser tattoo removal: laser principles and an updated guide for clinicians. *Laser Med Sci* **2022**, *37* (6), 2581-2587. DOI: 10.1007/s10103-022-03576-2.

(21) Mann, R.; Klingmuller, G. Electron-microscopic investigation of tattoos in rabbit skin. *Arch Dermatol Res* **1981**, *271* (4), 367-372. DOI: 10.1007/BF00406680.

(22) Baranska, A.; Shawket, A.; Jouve, M.; Baratin, M.; Malosse, C.; Voluzan, O.; Vu Manh, T.-P.; Fiore, F.; Bajénoff, M.; Benaroch, P.; et al. Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal. *Journal of Experimental Medicine* **2018**, *215* (4), 1115-1133. DOI: 10.1084/jem.20171608.

(23) Strandt, H.; Voluzan, O.; Niedermair, T.; Ritter, U.; Thalhamer, J.; Malissen, B.; Stoecklinger, A.; Henri, S. Macrophages and Fibroblasts Differentially Contribute to Tattoo Stability. *Dermatology* **2021**, *237* (2), 296-302. DOI: 10.1159/000506540.

(24) Yoon, S.; Kondakala, S.; Daddy-Gaoh, S.; Foley, S.; Kweon, O.; Kim, S. Causes, patterns, and epidemiology of tattoo-associated infections since 1820. *Lancet Microbe* **2025**, *6* (4), 7. DOI: 10.1016/j.lanmic.2024.101006.

(25) Kondakala, S.; Yoon, S.; Daddy-Gaoh, S.; Foley, S.; Kweon, O.; Kim, S. Tattoo-Associated Microbial Infections 1 Microbiology of tattoo-associated infections since 1820. *Lancet Microbe* **2025**, *6* (4), 7. DOI: 10.1016/j.lanmic.2024.101005.

(26) Morales-Cardona, C.; Llamas-Castellanos, B. Infectious complications of permanent tattoos: The skin out of law. Part I. *Iatreia* **2021**, *34* (4), 335-346. DOI: 10.17533/udea.iatreia.105.

(27) Sepehri, M.; Sejersen, T.; Qvortrup, K.; Lerche, C. M.; Serup, J. Tattoo Pigments Are Observed in the Kupffer Cells of the Liver Indicating Blood-Borne Distribution of Tattoo Ink. *Dermatology* **2017**, *233* (1), 86-93. DOI: 10.1159/000468149.

(28) Kolena, B.; Prochácková, N.; Hlisníková, H.; Nagyová, M.; Petrovicová, I. Harmful beauty-Temporal profile of urinary phthalate metabolites in tattooed volunteers from Slovakia. *Toxicol. Lett.* **2025**, *410*, 96-106. DOI: 10.1016/j.toxlet.2025.06.005.

(29) Laux, P.; Tralau, T.; Tentschert, J.; Blume, A.; Al Dahouk, S.; Baumler, W.; Bernstein, E.; Bocca, B.; Alimonti, A.; Colebrook, H.; et al. A medical-toxicological view of tattooing. *Lancet* **2016**, *387* (10016), 395-402. DOI: 10.1016/s0140-6736(15)60215-x.

(30) Humphries, A.; Lister, T. S.; Wright, P. A.; Hughes, M. P. Determination of the thermal and physical properties of black tattoo ink using compound analysis. *Lasers Med Sci* **2013**, *28* (4), 1107-1112. DOI: 10.1007/s10103-012-1198-9.

(31) Piccinini, P.; Pakalin, S.; Contor, L.; Bianchi, I. Safety of tattoos and permanent make-up Adverse health effects and experience with the Council of Europe Resolution (2008)1. *JRC Technical Reports* **2016**, *EUR 27672 EN*. DOI: 10.2788/177900.

(32) Petersen, H.; Lewe, D. Chemical purity and toxicology of pigments used in tattoo inks. *Curr Probl Dermatol* **2015**, *48*, 136-141. DOI: 10.1159/000369647.

(33) Commission, E. COMMISSION REGULATION (EU) amending Annex XVII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards substances in tattoo inks or permanent make-up. 2020; Vol. 2020/2081.

(34) Kröger, M.; Schleusener, J.; Lademann, J.; Meinke, M.; Jung, S.; Darvin, M. Tattoo pigments are localized intracellularly in the epidermis and dermis of fresh and old tattoos - in vivo study using two-photon excited FLIM. *Dermatology* **2023**, *239* (3), 478-493. DOI: 10.1159/000529577.
(35) Albanesi, C. Keratinocytes in allergic skin diseases. *Curr Opin Allergy Clin Immunol* **2010**, *10* (5), 452–456. DOI: 10.1097/ACI.0b013e32833e08ae.

(36) Dainese-Marque, O.; Garcia, V.; Andrieu-Abadie, N.; Riond, J. Contribution of Keratinocytes in Skin Cancer Initiation and Progression. *Int J Mol Sci* **2024**, *25* (16). DOI: 10.3390/ijms25168813.

(37) Juránová, J.; Franková, J.; Ulrichová, J. The role of keratinocytes in inflammation. *J Appl Biomed* **2017**, *15* (3), 169–179. DOI: 10.1016/j.jab.2017.05.003.

(38) Hogsberg, T.; Thomsen, B. M.; Serup, J. Histopathology and immune histochemistry of red tattoo reactions. Interface dermatitis is the lead pathology, with increase in T-lymphocytes and Langerhans cells suggesting an allergic pathomechanism. *Skin Res Technol* **2015**, *21* (4), 449–458. DOI: 10.1111/srt.12213.

(39) Aubry, L.; Vitipon, M.; Hirschler, A.; Diemer, H.; Rabilloud, T.; Carapito, C.; Douki, T. Accelerated aging modulates the toxicological properties of the diazo tattoo pigment PO13. *Scientific Reports* **2025**, *15* (1), 695. DOI: 10.1038/s41598-024-83713-9.

(40) Fraser, T. R.; Ross, K. E.; Alexander, U.; Lenehan, C. E. Current knowledge of the degradation products of tattoo pigments by sunlight, laser irradiation and metabolism: a systematic review. *Journal of Exposure Science & Environmental Epidemiology* **2022**, *32* (3), 343–355. DOI: 10.1038/s41370-021-00364-y.

(41) Aslan, C.; Gilbert, J. L. In Vitro Corrosion Assessment of the Essure(R) Medical Device in Saline, Simulated Inflammatory Solution and Neutral Buffered Formalin. *Acta Biomater* **2022**, *147*, 414–426. DOI: 10.1016/j.actbio.2022.05.028.

(42) Guldberg, M.; Christensen, V. R.; Perander, M.; Zoitos, B.; Koenig, A. R.; Sebastian, K. Measurement of in-vitro fibre dissolution rate at acidic pH. *Ann Occup Hyg* **1998**, *42* (4), 233–243. DOI: doi 10.1016/S0003-4878(98)00026-X.

(43) Stefaniak, A. B.; Guilmette, R. A.; Day, G. A.; Hoover, M. D.; Breysse, P. N.; Scripsick, R. C. Characterization of phagolysosomal simulant fluid for study of beryllium aerosol particle dissolution. *Toxicol In Vitro* **2005**, *19* (1), 123–134. DOI: 10.1016/j.tiv.2004.08.001.

(44) Valentine, R. C.; Shapiro, B. M.; Stadtman, E. R. Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. *Biochemistry* **1968**, *7* (6), 2143–2152. DOI: 10.1021/bi00846a017.

(45) Botelho, R. J.; Grinstein, S. Phagocytosis. *Curr Biol* **2011**, *21* (14), R533–538. DOI: 10.1016/j.cub.2011.05.053.

(46) Engel, E.; Vasold, R.; Santarelli, F.; Maisch, T.; Gopee, N. V.; Howard, P. C.; Landthaler, M.; Bäumler, W. Tattooing of skin results in transportation and light-induced decomposition of tattoo pigments – a first quantification in vivo using a mouse model. *Experimental Dermatology* **2010**, *19* (1), 54–60. DOI: 10.1111/j.1600-0625.2009.00925.x.

(47) Hauri, U.; Hohl, C. Photostability and breakdown products of pigments currently used in tattoo inks. *Current problems in dermatology* **2015**, *48*, 164–169. DOI: 10.1159/000369225.

(48) Scalia, S.; Dozzo, A.; Magli, S.; Scarcella, G. Incorporation in Lipid Microparticles of Acid Red 87, a Colorant Used in Tattoo Inks: Effect on Photodegradation Under Simulated Sunlight and Laser Radiation. *Photochemistry and Photobiology* **2020**, *96* (5), 998–1004. DOI: 10.1111/php.13258.

(49) Cui, Y. Y.; Spann, A. P.; Couch, L. H.; Gopee, N. V.; Evans, F. E.; Churchwell, M. I.; Williams, L. D.; Doerge, D. R.; Howard, P. C. Photodecomposition of Pigment Yellow 74, a pigment used in tattoo inks. *Photochemistry and Photobiology* **2004**, *80* (2), 175–184. DOI: 10.1562/2004-04-06-ra-136.1.

(50) Engel, E.; Spannberger, A.; Vasold, R.; König, B.; Landthaler, M.; Bäumler, W. Photochemical cleavage of a tattoo pigment by UVB radiation or natural sunlight. *Journal of the German Society of Dermatology* **2007**, *5* (7), 583–589. DOI: 10.1111/j.1610-0387.2007.06333.x.

(51) Bauer, E. M.; Ricci, C.; Cecchetti, D.; Ciufolini, G.; Cicero, D. O.; Rossi, M.; Guerriero, E.; Orlando, S.; Carbone, M. Toxicological problems of tattoo removal: characterization of femtosecond laser-induced fragments of Pigment Green 7 and Green Concentrate tattoo ink. *Arch Toxicol* **2025**, *99* (4), 1355–1369. DOI: 10.1007/s00204-024-03953-6.
(52) Schreiver, I.; Hutzler, C.; Laux, P.; Berlien, H.-P.; Luch, A. Formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. *Scientific Reports* **2015**, *5* (1), 12915. DOI: 10.1038/srep12915.

(53) Hering, H.; Sung, A. Y.; Röder, N.; Hutzler, C.; Berlien, H.-P.; Laux, P.; Luch, A.; Schreiver, I. Laser Irradiation of Organic Tattoo Pigments Releases Carcinogens with 3,3'-Dichlorobenzidine Inducing DNA Strand Breaks in Human Skin Cells. *The Journal of investigative dermatology* **2018**, *138* (12), 2687–2690. DOI: 10.1016/j.jid.2018.05.031.

(54) Anghelone, M.; Stoytschew, V.; Jembrih-Simbürger, D.; Schreiner, M. Spectroscopic methods for the identification and photostability study of red synthetic organic pigments in alkyd and acrylic paints. *Microchem J* **2018**, *139*, 155–163. DOI: 10.1016/j.microc.2018.02.029.

(55) Miao, C.; Jia, P. X.; Luo, C. N.; Pang, J. Y.; Xiao, L. Y.; Zhang, T. L.; Duan, J. C.; Li, Y.; Sun, Z. W. The size-dependent in vivo toxicity of amorphous silica nanoparticles: A systematic review. *Ecotox Environ Safe* **2024**, *271*. DOI: ARTN 115910

10.1016/j.ecoenv.2023.115910.

(56) Senut, M. C.; Zhang, Y. H.; Liu, F. C.; Sen, A.; Ruden, D. M.; Mao, G. Z. Size-Dependent Toxicity of Gold Nanoparticles on Human Embryonic Stem Cells and Their Neural Derivatives. *Small* **2016**, *12* (5), 631–646. DOI: 10.1002/smll.201502346.

(57) Sukhanova, A.; Bozrova, S.; Sokolov, P.; Berestovoy, M.; Karaulov, A.; Nabiev, I. Dependence of Nanoparticle Toxicity on Their Physical and Chemical Properties. *Nanoscale Res Lett* **2018**, *13*. DOI: ARTN 44

10.1186/s11671-018-2457-x.

(58) Rennick, J. J.; Johnston, A. P. R.; Parton, R. G. Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics. *Nat Nanotechnol* **2021**, *16* (3), 266–276. DOI: 10.1038/s41565-021-00858-8.

(59) Uribe-Querol, E.; Rosales, C. Phagocytosis: Our Current Understanding of a Universal Biological Process. *Front Immunol* **2020**, *11*, 1066. DOI: 10.3389/fimmu.2020.01066.

(60) Devcic, J.; Dussol, M.; Collin-Faure, V.; Perard, J.; Fenel, D.; Schoehn, G.; Carriere, M.; Rabilloud, T.; Dalzon, B. Immediate and Sustained Effects of Cobalt and Zinc-Containing Pigments on Macrophages. *Frontiers in Immunology* **2022**, *13*, 24. DOI: 10.3389/fimmu.2022.865239.

(61) Vitipon, M.; Akingbagbohun, E.; Rabilloud, T. The VVBlue assay: a plate-readable, dye exclusion-based cell viability assay for the toxicological testing of chemicals. *RSC Adv* **2025**, *15* (23), 17885–17896. DOI: 10.1039/d4ra08606f.

(62) Nguyen, J. A.; Yates, R. M. Better Together: Current Insights Into Phagosome-Lysosome Fusion. *Front Immunol* **2021**, *12*, 636078. DOI: 10.3389/fimmu.2021.636078.

(63) Sole-Domenech, S.; Singh, P. K.; Funes, L.; Ma, C. J.; Warren, J. D.; Maxfield, F. R. Real-time pH imaging of macrophage lysosomes using the pH-sensitive probe ApHID. *Cell Rep Methods* **2025**, *5* (10), 101203. DOI: 10.1016/j.crmeth.2025.101203.

(64) Galloway, C. J.; Dean, G. E.; Marsh, M.; Rudnick, G.; Mellman, I. Acidification of macrophage and fibroblast endocytic vesicles in vitro. *Proc Natl Acad Sci U S A* **1983**, *80* (11), 3334–3338. DOI: 10.1073/pnas.80.11.3334.

(65) Fels, A. O.; Nathan, C. F.; Cohn, Z. A. Hydrogen peroxide release by alveolar macrophages from sarcoid patients and by alveolar macrophages from normals after exposure to recombinant interferons alpha A, beta, and gamma and 1,25-dihydroxyvitamin D3. *J Clin Invest* **1987**, *80* (2), 381–386. DOI: 10.1172/JCI113083.

(66) Melo, R. C.; Fabrino, D. L.; D'Avila, H.; Teixeira, H. C.; Ferreira, A. P. Production of hydrogen peroxide by peripheral blood monocytes and specific macrophages during experimental infection with Trypanosoma cruzi in vivo. *Cell Biol Int* **2003**, *27* (10), 853–861. DOI: 10.1016/s1065-6995(03)00173-2.

(67) Nathan, C.; Nogueira, N.; Juangbhanich, C.; Ellis, J.; Cohn, Z. Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of Trypanosoma cruzi. *J Exp Med* **1979**, *149* (5), 1056–1068. DOI: 10.1084/jem.149.5.1056.
(68) Schneider, D. L. ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate. *J Biol Chem* **1983**, *258* (3), 1833-1838.

(69) Potts, G. D.; Jones, W.; Bullock, J. F.; Andrews, S. J.; Maginn, S. J. The Crystal-Structure of Quinacridone - an Archetypal Pigment. *J Chem Soc Chem Comm* **1994**, (22), 2565-2566. DOI: DOI 10.1039/c39940002565.

(70) Durán, N.; Silveira, C.; Durán, M.; Martinez, D. Silver nanoparticle protein corona and toxicity: a mini-review. *Journal of Nanobiotechnology* **2015**, *13*. DOI: 10.1186/s12951-015-0114-4.

(71) Lee, Y.; Choi, E.; Webster, T.; Kim, S.; Khang, D. Effect of the protein corona on nanoparticles for modulating cytotoxicity and immunotoxicity. *International Journal of Nanomedicine* **2015**, *10*, 97-112. DOI: 10.2147/ijn.S72998.

(72) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. *J Am Chem Soc* **2011**, *133* (8), 2525-2534. DOI: 10.1021/ja107583h.

(73) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. *Proc Natl Acad Sci U S A* **2008**, *105* (38), 14265-14270. DOI: 10.1073/pnas.0805135105.

(74) Yang, Y.; Han, P.; Xie, X.; Yin, X.; Duan, G.; Wen, L. Protein corona reduced graphene oxide cytotoxicity by inhibiting endocytosis. *Colloid Interface Sci. Commun.* **2021**, *45*, 6. DOI: 10.1016/j.colcom.2021.100514.

(75) de Oliveira, F.; Albuquerque, L.; Castro, C.; Riske, K.; Bellettini, I.; Giacomelli, F. Reduced cytotoxicity of nanomaterials driven by nano-bio interactions: Case study of single protein coronas enveloping polymersomes. *Colloid Surf. B-Biointerfaces* **2022**, *213*, 8. DOI: 10.1016/j colsurfb.2022.112387.

(76) Kihara, S.; Ashenden, A.; Kaur, M.; Glasson, J.; Ghosh, S.; van der Heijden, N.; Brooks, A. E. S.; Mata, J. P.; Holt, S.; Domigan, L. J.; et al. Cellular interactions with polystyrene nanoplastics-The role of particle size and protein corona. *Biointerphases* **2021**, *16* (4), 041001. DOI: 10.1116/6.0001124.

(77) Debnath, M.; Forster, J., 3rd; Ramesh, A.; Kulkarni, A. Protein Corona Formation on Lipid Nanoparticles Negatively Affects the NLRP3 Inflammasome Activation. *Bioconjug Chem* **2023**, *34* (10), 1766-1779. DOI: 10.1021/acs.bioconjchem.3c00329.

(78) Zhong, L.; Hu, X.; Cao, Z.; Wang, H.; Chen, Y.; Lian, H. Z. Aggregation and dissolution of engineering nano Ag and ZnO pretreated with natural organic matters in the simulated lung biological fluids. *Chemosphere* **2019**, *225*, 668-677. DOI: 10.1016/j.chemosphere.2019.03.080.

(79) Vranic, S.; Gosens, I.; Jacobsen, N. R.; Jensen, K. A.; Bokkers, B.; Kermanizadeh, A.; Stone, V.; Baeza-Squiban, A.; Cassee, F. R.; Tran, L.; et al. Impact of serum as a dispersion agent for in vitro and in vivo toxicological assessments of TiO(2) nanoparticles. *Arch Toxicol* **2017**, *91* (1), 353-363. DOI: 10.1007/s00204-016-1673-3.

(80) Escamilla-Rivera, V.; Uribe-Ramirez, M.; Gonzalez-Pozos, S.; Lozano, O.; Lucas, S.; De Vizcaya-Ruiz, A. Protein corona acts as a protective shield against Fe3O4-PEG inflammation and ROS-induced toxicity in human macrophages. *Toxicol Lett* **2016**, *240* (1), 172-184. DOI: 10.1016/j.toxlet.2015.10.018.

(81) Liu, N.; Liang, Y.; Wei, T.; Zou, L.; Bai, C.; Huang, X.; Wu, T.; Xue, Y.; Tang, M.; Zhang, T. Protein corona mitigated the cytotoxicity of CdTe QDs to macrophages by targeting mitochondria. *NanolImpact* **2022**, *25*, 100367. DOI: 10.1016/j.impact.2021.100367.

(82) Ge, C.; Du, J.; Zhao, L.; Wang, L.; Liu, Y.; Li, D.; Yang, Y.; Zhou, R.; Zhao, Y.; Chai, Z.; et al. Binding of blood proteins to carbon nanotubes reduces cytotoxicity. *Proc Natl Acad Sci U S A* **2011**, *108* (41), 16968-16973. DOI: 10.1073/pnas.1105270108.

(83) Shinohara, N.; Zhang, G.; Oshima, Y.; Kobayashi, T.; Imatanaka, N.; Nakai, M.; Sasaki, T.; Kawaguchi, K.; Gamo, M. Kinetics and dissolution of intratracheally administered nickel oxide nanomaterials in rats. *Part Fibre Toxicol* **2017**, *14* (1), 48. DOI: 10.1186/s12989-017-0229-x.

(84) Lecuyer, T.; Durand, M. A.; Volatron, J.; Desmau, M.; Lai-Kuen, R.; Corvis, Y.; Seguin, J.; Wang, G.; Alloyeau, D.; Scherman, D.; et al. Degradation of ZnGa(2)O(4):Cr(3+) luminescent nanoparticles in lysosomal-like medium. *Nanoscale* **2020**, *12* (3), 1967-1974. DOI: 10.1039/c9nr06867h.
(85) Schiavo, B.; Morton-Bermea, O.; Meza-Figueroa, D.; Acosta-Elias, M.; Gonzalez-Grijalva, B.; Armienta-Hernandez, M. A.; Inguaggiato, C.; Valera-Fernandez, D. Characterization and Polydispersity of Volcanic Ash Nanoparticles in Synthetic Lung Fluid. *Toxics* **2023**, *11* (7). DOI: 10.3390/toxics11070624.

(86) Soran, A.; Kanbour-Shakir, A.; Bas, O.; Bonaventura, M. A tattoo pigmented node and breast cancer. *Bratisl Lek Listy* **2014**, *115* (5), 311–312. DOI: 10.4149/bll_2014_063.

(87) Yactor, A. R.; Michell, M. N.; Koch, M. S.; Leete, T. G.; Shah, Z. A.; Carter, B. W. Percutaneous tattoo pigment simulating calcific deposits in axillary lymph nodes. *Proc (Bayl Univ Med Cent)* **2013**, *26* (1), 28–29. DOI: 10.1080/08998280.2013.11928907.

(88) Reddersen, K.; Gregersen, D. M.; Tittelbach, J.; Wiegand, C. Tattooed human in vitro skin model for testing the biocompatibility of tattoo inks and healing progression after tattooing. *Sci Rep* **2025**, *15* (1), 2277. DOI: 10.1038/s41598-025-86813-2.
Modulation of the toxicity of three red tattoo
pigments by artificial aging

Lise Aubry†, Marie Blanchemanche†, Lucie Breuillé†, Thierry Rabilloud‡, and Thierry Douki†,‡

† Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, IRIG, SyMMES, F-38000 Grenoble,
France

‡ Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG
LCBM, F 38054 Grenoble, France

**Supporting Information**

* Corresponding author:

Thierry Douki

SyMMES UMR 5819, CEA – UGA - CNRS - Grenoble-INP, DRF/IRIG, CEA-Grenoble, 17
Avenue des Martyrs, 38054 Grenoble Cedex 9, France; [orcid.org/0000-0002-5022-071X](https://orcid.org/0000-0002-5022-071X)

Email: thierry.douki@cea.fr
Table des matières

<table><tr><td>Table S1: Zeta potential of PR254 and PR122 particles in control and photoaged samples.</td><td>S44</td></tr><tr><td>Table S2: Loss of viability of HaCaT cells exposed to increasing concentrations of filtrates of pigment suspension arising from either control or PGL samples.</td><td>S44</td></tr><tr><td>Figure S1: Induction of ROS in HaCaT cells exposed to control or photoaged PR254 suspensions. Cells were exposed to PR254 for 4 hours.</td><td>S45</td></tr><tr><td>Figure S2: Impact of photoaging on the size distribution of PR122 suspensions.</td><td>S45</td></tr><tr><td>Figure S3: Characterization of PR122 photoproduct PPD1 (11-hydroxy-8-methyl-2,3-dihydropyrido[2,3-b]acridin-4(1H)-one).</td><td>S46</td></tr><tr><td>Figure S4: Characterization of PR122 photoproduct PPD2 4-amino-2-(hydroxy(p-tolyl)methyl)butanoic acid).</td><td>S47</td></tr><tr><td>Figure S5: Characterization of PR122 photoproduct PPD3 ((E)-3-(2-aminovinyl)-7-methyl-2-vinyl-1,4-dihydroquinolin-4-ol).</td><td>S48</td></tr><tr><td>Figure S6: Characterization of PR122 photoproduct PPD4 (2-amino-1,4,9-trihydroxy-6-methylacridin-3-yl)(p-tolyl)methanone).</td><td>S49</td></tr><tr><td>Figure S7: Characterization of PR122 photoproduct PPD5 (9-hydroxy-6-methylene-6,10-dihydroacridin-2(4aH)-one).</td><td>S50</td></tr><tr><td>Figure S8: Characterization of PR122 photoproduct PPD6, hydrated derivative of PPD4.</td><td>51</td></tr><tr><td>Figure S9: Detection and characterization of photoproduct PPD2 and PPD5 in an isopropanol extract of suspensions of PR122 photoaged in water.</td><td>S52</td></tr><tr><td>Figure S10: Detection and characterization of the two isomers of photoproduct PPD6 in an isopropanol extract of suspensions of PR122 photoaged in water.</td><td>S53</td></tr><tr><td>Figure S11: Detection and characterization of photoproduct PPD2 in an aqueous extract of suspensions of PR122 photoaged in water.</td><td>S53</td></tr><tr><td>Figure S12: Impact of photoaging on cytotoxicity of PR122 in HaCaT cells.</td><td>S54</td></tr><tr><td>Figure S13: Picture of PGL suspensions of PR122 after 10 days of incubation in complete phagolysosomal-mimicking medium (PGL), in PGL medium without Ig (PGL w/o Ig), and in water.</td><td>S55</td></tr></table>
<table><caption>**Table S1:** Zeta potential of PR254 and PR122 particles in control and photoaged samples. Values, expressed in mV, were determined by electrophoretic light scattering.</caption><thead><tr><th>Pigment</th><th>Condition</th><th>Zeta potential (mV)</th></tr></thead><tbody><tr><td rowspan="3">PR254</td><td>Pristine</td><td>-44.7</td></tr><tr><td>Temperature control (96h)</td><td>-48.7</td></tr><tr><td>Photoaged (96h)</td><td>-43.6</td></tr><tr><td rowspan="3">PR122</td><td>Pristine</td><td>-29.9</td></tr><tr><td>Temperature control (96h)</td><td>-17.7</td></tr><tr><td>Photoaged (96h)</td><td>-21.0</td></tr></tbody></table>

<table><caption>**Table S2:** Loss of viability of HaCaT cells exposed to increasing concentrations of filtrates of pigment suspension arising from either control or PGL samples. Cells were exposed to the filtrates of pigment suspension (µg mL⁻¹) for 24h. Viability was assessed by the MTT assay. Values are expressed as mean cell viability ± standard deviation (n=6). The reported statistical analysis was applied to the difference between samples incubated in water or in PGL for a specific pigment at a specific concentration (*p* ≤ 0.05)</caption><tbody><tr><td colspan="6">PR254</td></tr><tr><td>Control 0.03</td><td>PGL 0.03</td><td>Control 0.3</td><td>PGL 0.3</td><td>Control 3</td><td>PGL 3</td></tr><tr><td>96.0±16.2</td><td>103.4±10.1</td><td>90.9±30.2</td><td>95.6±30.2</td><td>75.7±29.8</td><td>87.2±19.3</td></tr><tr><td colspan="6">PR122</td></tr><tr><td>Control 0.03</td><td>PGL 0.03</td><td>Control 0.3</td><td>PGL 0.3</td><td>Control 1</td><td>PGL 1</td></tr><tr><td>100.8±5.8</td><td>96.5±5.8</td><td>88.4±17.1</td><td>95.2±7.8</td><td>77.7±12.2</td><td>90.1±5.7 *</td></tr><tr><td colspan="6">PO13</td></tr><tr><td>Control 0.3</td><td>PGL 0.3</td><td>Control 3</td><td>PGL 3</td><td>Control 30</td><td>PGL 30</td></tr><tr><td>72.8±8.4</td><td>100.9±5.5</td><td>57.1±7.7</td><td>91.5±8.1</td><td>55.1±6.5</td><td>76.1±7.2</td></tr></tbody></table>
**Figure S1:** Induction of ROS in HaCaT cells exposed to control or photoaged PR254 suspensions. Cells were exposed to PR254 for 4 hours. ROS production was assessed by using the CM-H₂DCFDA assay. The results represent normalized fluorescence intensities and are expressed as the medians ± 2nd and 3rd quartiles, with maximum and minimum (n=9). tBuOOH was used as a positive control. $\$ indicates significant differences from vehicle control (Kruskal-Wallis test followed by Dunn's multiple comparisons, $$$ p \le 0.0001). Kolmogorov-Smirnov test was also performed and no difference between photoaged PR254 and temperature control at the same concentration of pigment was found.

<figure><img src="image_12.png" /></figure>

**Figure S2:** Impact of photoaging on the size distribution of PR122 suspensions. The hydrodynamic diameter was measured by DLS. No statistically significant difference was found between the pristine, the control and the photoaged suspensions.

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>**Figure S3:** Characterization of PR122 photoproduct PPD1 (11-hydroxy-8-methyl-2,3-dihydropyrido[2,3-b]acridin-4(1H)-one) : a) proposed structure and mechanism of formation, b) MS1 mass spectrum, c) UV-visible absorption spectrum, d) fragmentation mass spectrum MS2 at high collision energy (40 mV), e) fragmentation mass spectrum MS2 at low collision energy (25 mV), and f) proposed fragmentation pathway</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure S4:** Characterization of PR122 photoproduct PPD2 4-amino-2-(hydroxy(*p*-tolyl)methyl)butanoic acid): a) proposed structure and mechanism of formation, b) MS1 mass spectrum, c) UV-visible absorption spectrum, d) fragmentation mass spectrum MS2 at high collision energy (40 mV), e) fragmentation mass spectrum MS2 at low collision energy (25 mV), and f) proposed fragmentation pathway</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure S5:** Characterization of PR122 photoproduct PPD3 ((E)-3-(2-aminovinyl)-7-methyl-2-vinyl-1,4-dihydroquinolin-4-ol): a) proposed structure and mechanism of formation, b) MS1 mass spectrum, c) UV-visible absorption spectrum, d) fragmentation mass spectrum MS2 at high collision energy (40 mV), e) fragmentation mass spectrum MS2 at low collision energy (25 mV), and f) proposed fragmentation pathway</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Figure S6:** Characterization of PR122 photoproduct PPD4 (2-amino-1,4,9-trihydroxy-6-methylacridin-3-yl)(p-tolyl)methanone): a) proposed structure and mechanism of formation, b) MS1 mass spectrum, c) UV-visible absorption spectrum, d) fragmentation mass spectrum MS2 at high collision energy (40 mV), e) fragmentation mass spectrum MS2 at low collision energy (25 mV), and f) proposed fragmentation pathway.</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>**Figure S7:** Characterization of PR122 photoproduct PPD5 (9-hydroxy-6-methylene-6,10-dihydroacridin-2(4aH)-one): a) proposed structure and mechanism of formation, b) MS1 mass spectrum, c) UV-visible absorption spectrum, d) fragmentation mass spectrum MS2 at high collision energy (40 mV), e) fragmentation mass spectrum MS2 at low collision energy (25 mV), and f) proposed fragmentation pathway.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure S8:** Characterization of PR122 photoproduct PPD6, hydrated derivative of PPD4. Its exact structure remains to be fully elucidated. a) MS1 mass spectrum, b) UV-visible absorption spectrum, c) fragmentation mass spectrum MS2 at high collision energy (40 mV), and d) fragmentation mass spectrum MS2 at low collision energy (25 mV).</figcaption></figure>
**Figure S9:** Detection and characterization of photoproduct PPD2 and PPD5 in an isopropanol extract of suspensions of PR122 photoaged in water. a) HPLC chromatograms monitored in production ion scan mode with the pseudo-molecular ions of the six identified PR122 photoproducts. The peak at 16.6 min is a solvent impurity. b) MS2 mass spectrum (collision energy 30 mV) of the peak at 15.5 min (PPD2) and c) MS2 mass spectrum (collision energy 30 mV) of the peak at 18.7 min (PPD5). These two fragmentation spectra are identical to those of photoproducts isolated upon photoaging of PR122 in isopropanol.

<figure><img src="image_20.png" /></figure>
**Figure S10:** Detection and characterization of the two isomers of photoproduct PPD6 in an isopropanol extract of suspensions of PR122 photoaged in water. a) HPLC chromatograms of the fragment at m/z 281 upon detection in production ion scan mode of the pseudo-molecular ion at m/z 393. b) MS2 mass spectrum of the peaks eluted between 15.0 and 15.5 min (collision energy 30 mV).

<figure><img src="image_21.png" /></figure>

**Figure S11:** Detection and characterization of photoproduct PPD2 in an aqueous extract of suspensions of PR122 photoaged in water. a) HPLC chromatograms monitored in production ion scan mode with the pseudo-molecular ions of the six identified PR122 photoproducts. The peaks at 14.5 and 16.6 min are impurities present in the control. b) MS2 mass spectrum (collision energy 30 mV) of the peak eluted at 15.5 min (PPD2).

<figure><img src="image_22.png" /></figure>
**Figure S12:** Impact of photoaging on cytotoxicity of PR122 in HaCaT cells: Viability was assessed by the MTT assay. a) cells were exposed to increasing concentration of suspension and b) cells were exposed to increasing amounts of filtrate prepared from the suspensions. After 24h, cell viability was assessed by the MTT assay. The results are represented by the medians of normalized cell viability ± 2nd and 3rd quartiles, with maximum and minimum. $ indicates significant differences from vehicle control (Kruskal-Wallis test followed by Dunn's multiple comparisons, $ p ≤ 0.05, $$ p ≤ 0.01). * indicates significant differences between photoaged PR122 and temperature control at the same concentration of pigment (Kolmogorov-Smirnov test, * p ≤ 0.05).

<figure><img src="image_23.png" /><figcaption>b) filtrate concentration (µg mL⁻¹)</figcaption></figure>

<figure><img src="image_24.png" /></figure>
<figure><img src="image_25.png" /><figcaption>**Figure S13:** Picture of PGL suspensions of PR122 after 10 days of incubation in complete phagolysosomal-mimicking medium (PGL), in PGL medium without Ig (PGL w/o Ig), and in water.</figcaption></figure>