Applicability of In Silico New Approach Methods for the Risk Assessment of Tattoo Ink Ingredients.

Nowadays, about 12% of the European and 20% of the US population are tattooed. Rising concerns regarding consumer safety, led to legal restrictions on tattoo and permanent make-up (PMU) inks. Restrictions also include bans on certain colourants. Both ink types use organic pigments for colour-giving, plus inorganic pigments for white and black and colour tones. Pigments are only sparingly soluble in common solvents and occur as suspended particles in the ink matrix. Their detection and identification therefore pose a major challenge for laboratories involved in monitoring the legal compliance of tattoo inks and PMU. We overcame this challenge by developing a direct laser desorption ionisation time-of-flight mass spectrometry method, which included an easy sample clean up. The method proved to be capable of detecting and identifying organic pigments in almost all of the tested ink samples. Method validation and routine deployment during market surveys showed the method to be fit for purpose. Pigment screening of 396 tattoo inks and 55 PMU taken from the Swiss market between 2009 and 2017 lead to the following conclusions: Pigment variety is much greater in tattoo inks (18) than in PMU (10); four prohibited pigments (Pigment Green 7, Pigment Red 122, Pigment Violet 19 and 23) were found in both ink types; for PMU, these four pigments made up 12% of the pigment findings, compared to 32% for tattoo inks. Therefore, legal compliance of PMU was at a higher level. A comparison of pigments found with those declared on tattoo ink labels clearly showed that banned pigments are rarely declared, but rather masked by listing non present legal pigments and label forging; therefore, highlighting the urgency of widespread market controls.

Nowadays, about 12% of the European and 20% of the US population are tattooed. Rising concerns regarding consumer safety, led to legal restrictions on tattoo and permanent make-up (PMU) inks. Restrictions also include bans on certain colourants. Both ink types use organic pigments for colour-giving, plus inorganic pigments for white and black and colour tones. Pigments are only sparingly soluble in common solvents and occur as suspended particles in the ink matrix. Their detection and identification therefore pose a major challenge for laboratories involved in monitoring the legal compliance of tattoo inks and PMU. We overcame this challenge by developing a direct laser desorption ionisation time-of-flight mass spectrometry method, which included an easy sample clean up. The method proved to be capable of detecting and identifying organic pigments in almost all of the tested ink samples. Method validation and routine deployment during market surveys showed the method to be fit for purpose. Pigment screening of 396 tattoo inks and 55 PMU taken from the Swiss market between 2009 and 2017 lead to the following conclusions: Pigment variety is much greater in tattoo inks (18) than in PMU (10); four prohibited pigments (Pigment Green 7, Pigment Red 122, Pigment Violet 19 and 23) were found in both ink types; for PMU, these four pigments made up 12% of the pigment findings, compared to 32% for tattoo inks. Therefore, legal compliance of PMU was at a higher level. A comparison of pigments found with those declared on tattoo ink labels clearly showed that banned pigments are rarely declared, but rather masked by listing non present legal pigments and label forging; therefore, highlighting the urgency of widespread market controls.

Today's tattoo inks are no longer just simple solids in liquid suspension. Nowadays, these inks are high-tech dispersions made from finely spread pigments in a binder-solvent mixture. These so-called colour dispersions must follow the modern standards of tattooing, which are increasing every year. They must be rich in chromophoric pigments and yet fluid, they must not dry rapidly, and there should be no occurrence of any sedimentation, even during longer tattoo seasons. An innovative tattoo ink should enable long-lasting, brilliant tattoos without a negative impact on the artist's workflow and of course without endangering the consumer. The high standard in tattoos, regarding the motives and techniques, that is witnessed today could not be achieved by the artists without quality tools and modern tattoo ink. This article will give the reader a brief overview of the different ingredients of tattoo ink and of the function of binding agents and solvents in modern tattoo ink as well as describe what additives are used to achieve the desired behaviour during application. Furthermore, the article will take a look into the pigments that are used in tattoo ink and show why certain pigments are not suited for tattoo ink. The differences, advantages and disadvantages of organic and inorganic pigments will be explained.

Since 2020, the REACh regulation requires toxicological data on nanoforms of materials, including the assessment of their skin-sensitizing properties. Small molecules' skin sensitization potential can be assessed by new approach methodologies (NAMs) addressing three key events (KE: protein interaction, activation of dendritic cells, and activation of keratinocytes) combined in a defined approach (DA) described in the OECD guideline 497. In the present study, the applicability of three NAMs (DPRA, LuSens, and h-CLAT) to nine materials (eight inorganic nanomaterials (NM) consisting of CeO2, BaSO4, TiO2 or SiO2, and quartz) was evaluated. The NAMs were technically applicable to NM using a specific sample preparation (NANOGENOTOX dispersion protocol) and method modifications to reduce interaction of NM with the photometric and flowcytometric read-outs. The results of the three assays were combined according to the defined approach described in the OECD guideline No. 497; two of the inorganic NM were identified as skin sensitizers. However, data from animal studies (for ZnO, also human data) indicate no skin sensitization potential. The remaining seven test substances were assessed as "inconclusive" because all inorganic NM were outside the domain of the DPRA, and the achievable test concentrations were not sufficiently high according to the current test guidelines of all three NAMs. The use of these NAMs for (inorganic) NM and the relevance of the results in general are challenged in three ways: (i) NAMs need modification to be applicable to insoluble, inorganic matter; (ii) current test guidelines lack adequate concentration metrics and top concentrations achievable for NM; and (iii) NM may not cause skin sensitization by the same molecular and cellular key events as small organic molecules do; in fact, T-cell-mediated hypersensitivity may not be the most relevant reaction of the immune system to NM. We conclude that the NAMs adopted by OECD test guidelines are currently not a good fit for testing inorganic NM.

Tattoo inks have been reported to elicit allergic contact dermatitis. To investigate the labels and the contents of metals and pigments in tattoo inks, considering restrictions within the European Union. Seventy-three tattoo inks currently available on the market, either bought or donated (already used), were investigated for trace metals and pigments by inductively coupled plasma mass spectrometry and by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry. Ninety-three percent of the bought tattoo inks violated European, legal requirements on labeling. Fifty percent of the tattoo inks declared at least one pigment ingredient incorrectly. Sixty-one percent of the inks contained pigments of concern, especially red inks. Iron, aluminium, titanium, and copper (most in green/blue inks) were the main metals detected in the inks. The level of metal impurities exceeded current restriction limits in only a few cases. Total chromium (0.35-139 μg/g) and nickel (0.1-41 μg/g) were found in almost all samples. The levels of iron, chromium, manganese, cobalt, nickel, zinc, lead, and arsenic were found to covary significantly. To prevent contact allergy and toxic reactions among users it is important for tattoo ink manufacturers to follow the regulations and decrease nickel and chromium impurities.

Novel techniques and methodologies are being developed to advance food safety risk assessment into the next-generation. Considering the shortcomings of traditional animal testing, new approach methodologies (NAMs) will be the main tools for the next-generation risk assessment (NGRA), using non-animal methodologies such as in vitro and in silico approaches. The United States Environmental Protection Agency and the European Food Safety Authority have established work plans to encourage the development and application of NAMs in NGRA. Currently, NAMs are more commonly used in research than in regulatory risk assessment. China is also developing NAMs for NGRA but without a comprehensive review of the current work. This review summarizes major NAM-related research articles from China and highlights the China National Center for Food Safety Risk Assessment (CFSA) as the primary institution leading the implementation of NAMs in NGRA in China. The projects of CFSA on NAMs such as the Food Toxicology Program and the strategies for implementing NAMs in NGRA are outlined. Key issues and recommendations, such as discipline development and team building, are also presented to promote NAMs development in China and worldwide.

Nowadays, about 12% of the European and 20% of the US population are tattooed. Rising concerns regarding consumer safety, led to legal restrictions on tattoo inks and permanent make-up (PMU) inks. Restrictions also include bans on certain hazardous colourants. Both ink types use organic pigments for colour-giving, plus inorganic pigments for white and black and colour tones. Pigments are only sparingly soluble in common solvents and occur as suspended particles in the ink matrix. Their detection and identification therefore pose a major challenge for laboratories involved in monitoring the legal compliance of tattoo inks and PMUs. We overcame this challenge by developing a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry method, which included an easy sample clean up. The method proved to be capable of detecting and identifying organic pigments in almost all of the tested ink samples. Method validation and routine deployment during market surveys showed the method to be fit for purpose. Pigment screening of 396 tattoo inks and 55 PMUs taken from the Swiss market between 2009 and 2017 lead to the following conclusions: Pigment variety is much greater in tattoo inks (18) than in PMUs (10); four prohibited pigments (Pigment Green 7, Pigment Red 122, Pigment Violet 19 and 23) were found in both ink types; for PMUs, these four pigments made up 12% of the pigment findings, compared to 32% for tattoo inks. Therefore, legal compliance of PMUs was at a higher level. A comparison of pigments found with those declared on tattoo ink labels clearly showed that banned pigments are rarely declared, but rather masked by listing not present legal pigments and label forging; therefore, highlighting the urgency of widespread market controls.

Robust and efficient processes are needed to establish scientific confidence in new approach methodologies (NAMs) if they are to be considered for regulatory applications. NAMs need to be fit for purpose, reliable and, for the assessment of human health effects, provide information relevant to human biology. They must also be independently reviewed and transparently communicated. Ideally, NAM developers should communicate with stakeholders such as regulators and industry to identify the question(s), and specified purpose that the NAM is intended to address, and the context in which it will be used. Assessment of the biological relevance of the NAM should focus on its alignment with human biology, mechanistic understanding, and ability to provide information that leads to health protective decisions, rather than solely comparing NAM-based chemical testing results with those from traditional animal test methods. However, when NAM results are compared to historical animal test results, the variability observed within animal test method results should be used to inform performance benchmarks. Building on previous efforts, this paper proposes a framework comprising five essential elements to establish scientific confidence in NAMs for regulatory use: fitness for purpose, human biological relevance, technical characterization, data integrity and transparency, and independent review. Universal uptake of this framework would facilitate the timely development and use of NAMs by the international community. While this paper focuses on NAMs for assessing human health effects of pesticides and industrial chemicals, many of the suggested elements are expected to apply to other types of chemicals and to ecotoxicological effect assessments.

A new high-throughput method to make a quality control on tattoo inks.

It is estimated that more than 60 million people in Europe, that is, around 12% of the European population, have at least one tattoo. However, there is still little information on the long-term effects of tattoos. Inks used for tattooing are a mixture of chemicals, with pigments being the main components responsible for the visual effect. The pigments used are not produced specifically as ingredients for tattooing, but mainly/primarily for the needs of industry, where lower purity requirements and quality standards are acceptable. It is therefore necessary to understand the risks associated with tattoos, but also to implement appropriate legal regulations. The aim of this article was to collect and summarise the results of research conducted so far on the type of colourants used in tattoo ink and to analyze the impact of these on human health. In addition, as part of this work, the current legal acts regulating the concentration limits and composition of inks used in tattooing as well as the psychological aspects of tattooing were collected and presented. Scientific reports and articles from renowned journals from 1994 to 2022, relevant review and research publications in PubMed, and Google Scholar were analyzed. To analyze the available research literature, the Web of Science, Scopus, PubMed databases were used. The following keywords were used to search for publications: tattoos, colourants used in tattoos, side effects of tattoos, legal acts, psychological aspects of tattoos. The result of the literature analysis indicates a risk to health and side effects associated with tattooing the body. There are still no standardised test methods to analyze tattoo inks and assess their safety. Although the art of tattooing has been known for millennia, European legal authorities have not yet implemented effective regulations. Currently, tattoo products in Europe are covered by the general REACH regulation (Resolution ResAP, 2008; EU regulation 2020/2081, 2020). on product safety. The new amendment in force since January 4, 2022 introduces concentration limits for certain substances used in tattoo and permanent makeup inks. However, these provisions do not sufficiently protect either the consumer or the tattoo industry. The results of the research indicate a potentially harmful effect on skin health. A more stringent safety assessment of the colourants used for tattooing is recommended, supported by studies and applicable legislation.

The growing popularity of tattoos highlights the importance of careful regulation of tattoo inks to minimise health risks. In Switzerland, tattoo inks are subject to strict legal requirements based on food legislation, which are designed to ensure that they are sterile and harmless. The European Union has significantly tightened restrictions on substances in tattoo inks through Regulation (EU) 2020/2081 in order to increase safety. In contrast, the USA regulate tattoo inks under the supervision of the Food and Drug Administration (FDA), but do not have specific pre-market controls for tattoo ink products. This comparison highlights different regulatory approaches in Switzerland, the EU and the USA and underlines the need for more harmonised global regulation to protect consumer health.

The increasing prevalence of tattoos and the frequent occurrence of adverse skin reactions, particularly from colored inks, highlight the urgent need for reliable analytical methods to identify potentially harmful substances in tattoo inks and tattooed skin. In this study, a novel, laser-based technique enabling combined molecular and elemental analysis is presented: Laser Ablation hyphenated to Atmospheric Pressure Chemical Ionization-Mass Spectrometry (LA-APCI-MS) and Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS/APCI-MS). This instrumental setup allows the simultaneous detection of organic and inorganic pigments as well as their metallic contaminants directly in solid samples. A newly developed spectral pigment libraries based on MS1 and MS2 data enables precise identification of organic pigments within complex matrices such as inks or skin sections. Using five commercially available tattoo inks, this new technique reliably identified nondeclared or even prohibited organic pigments and different metals. In skin biopsies from red tattoos with adverse reactions, different organic pigments and metals were localized via bioimaging, revealing distribution patterns that may help to uncover the causes of allergic responses. The introduced LA-ICP-MS/APCI-MS technique offers a rapid and powerful approach to obtain complementary information on organic pigments, inorganic compounds, and metals from a single sample. As such, it represents a significant advancement for investigating tattoo-related side effects, supporting risk assessment, and ensuring regulatory compliance.

New approach methodologies (NAMs), which include in vitro, in silico, and other non-animal methods of testing, are poised to revolutionize the traditional preclinical safety assessment paradigms. There has been growing support for NAMs by the regulatory authorities globally, due to ethical, scientific, and policy imperatives to reduce the use of animals in testing. Yet, all enthusiasm around the potential of NAMs notwithstanding, there lingers one crucial question: Does early regulatory engagement on NAM accelerate the preclinical development timelines? It is hoped that this review brings us to the state of existing literature and regulatory frameworks to appraise the impact of early dialogue with Regulatory agencies such as the FDA, EMA, Health Canada, and PMDA on development trajectories. The article looks at the timelines involved with traditional versus NAM-based preclinical approaches, the efficiency of early scientific advice procedures, and the obstacles to full adoption. Main focus is laid on the timing of early engagement strategies (e.g., pre-IND and Scientific Advice meetings) as potential key thinking points to reduce the overall development time and to allow NAMs to be accepted and validated earlier in the drug development process and thus streamline the regulatory submissions. The review concludes that while early engagement enhances regulatory clarity, its ability to accelerate timelines depends on key factors. These include regulatory agency familiarity with NAMs, data standardization, and the harmonization of international expectations.

Tattooing is an ancient practice and its popularity has been increasing in the recent years. After tattooing, complications may occur related to compose tattoo inks. In this study, the phototoxicity potential of the blue, red and black colors of the most commonly used three different commercially-available tattoo ink brands have been examined by performing in vitro 3T3-neutral red uptake (NRU) phototoxicity test. In the study, the phototoxicity of serial diluted concentrations of tattoo inks were evaluated with in vitro 3T3-NRU phototoxicity test method according to OECD guide 432. The data obtained from the NRU test result were uploaded to Phototox software (version 2.0) and the phototoxicity potentials of tattoo inks were determined via the calculation of the mean photo effect (MPE) and photo irritation factor (PIF) values. The red, black and blue colors of three different commercially available tattoo inks did not cause a cytotoxic activity on BALB/c 3T3 cells with 3T3-NRU test. The IC50 values could not be determined +ultraviolet (UV) and -UV conditions. PIF values could not be calculated and MPE values were <0.1, which predicts the absence of phototoxic effect for all of the tested tattoo inks. All tested inks were evaluated as non-phototoxic according to the results of MPE values calculated using Phototox software. However, test results should be verified by other phototoxicity test methods to obtain a comprehensive evaluation of phototoxic complications of different tattoo inks.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods’ predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.