Development of a human liver microphysiological co-culture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites and exhibit altered toxicity compared to their parent compounds. This paper describes a two-chamber liver-organ co-culture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This two-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a two-dimensional (2D) cell mono-layer. Culture medium and compounds freely diffuse between the two chambers. Human differentiated HepaRGTM liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 (CYP3A4) enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this co-culture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ co-culture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals, to better recapitulate the biological effects and potential toxicity of human exposures.

Method validation and environmental monitoring of triethanolamine ester quaternary ammonium compounds.

In this study water and sediment samples, collected from the River Nene (Northamptonshire) at several sites in the vicinity of the Great Billing sewage treatment plant (STP), were analysed for triethanolamine quaternary compounds (TEAQ, ester quats). A method was developed using liquid chromatography tandem mass spectrometry (LC/MS/MS) with a electrospray ionisation source (ESI). Ten components were determined using a characterised commercial sample of Tallow TEAQ as a standard. To our knowledge this is the first time environmental concentrations of a wide spectrum of individual homologues of TEAQ have been reliably quantified covering a broad range of environmental matrices (STP influent, STP effluent, surface waters and sediments), due to the challenging nature of the analytical method. The method featured novel solutions for the determination of long and multiple chain length alkyl quats, controlling loss processes, background contamination and chromatographic performance. TEAQ compounds were found to be highly removed in the sewage treatment plant resulting in low effluent concentrations. Low concentrations in both river water and sediment samples were found also. In many cases levels were below the Method Detection Limit (MDL). In river water samples, mean values of TEAQ compounds found were 210-398 ng/L for C16:0/C18:0 TEAQ diester and 126-287 ng/L for C18:0/C18:0 TEAQ diester. River sediment was found to contain mean TEAQ levels of 7.07-12.5, 19.7 to 40.3 and 7.04-35.1 µg/kg dry weight for C16:0/C16:0, C16:0/C18:0, and C18:0/C18:0 TEAQ, respectively. At Great Billing STP monoesters and diesters of TEAQ were shown to be efficiently removed (>97 and 99 %, respectively), although limited samples were taken on this occasion.

ADME characterization and PBK model development of 3 highly protein-bound UV filters through topical application.

Estimating human exposure in the safety assessment of chemicals is crucial. Physiologically based kinetic (PBK) models which combine information on exposure, physiology, and chemical properties, describing the absorption, distribution, metabolism, and excretion (ADME) processes of a chemical, can be used to calculate internal exposure metrics such as maximum concentration and area under the concentration-time curve in plasma or tissues of a test chemical in next-generation risk assessment. This article demonstrates the development of PBK models for 3 UV filters, specifically octyl methoxycinnamate, octocrylene, and 4-methylbenzylidene camphor. The models were parameterized entirely based on data obtained from in vitro and/or in silico methods in a bottom-up modeling approach and then validated based on human dermal pharmacokinetic (PK) data. The 3 UV filters are "difficult to test" in in vitro test systems due to high lipophilicity, high binding affinity for proteins, and nonspecific binding, for example, toward plastic. This research work presents critical considerations in ADME data generation, interpretation, and parameterization to assure valid PBK model development to increase confidence in using PBK modeling to help make safety decisions in the absence of human PK data. The developed PBK models of the 3 chemicals successfully simulated the plasma concentration profiles of clinical PK data following dermal application, indicating the reliability of the ADME data generated and the parameters determined. The study also provides insights and lessons learned for characterizing ADME and developing PBK models for highly lipophilic and protein-bound chemicals in the future.

PBK modelling of topical application and characterisation of the uncertainty of Cmax estimate: A case study approach.

Combined with in vitro bioactivity data, physiologically based kinetic (PBK) models has increasing applications in next generation risk assessment for animal-free safety decision making. A tiered framework of building PBK models for such application has been developed with increasing complexity and refinements, as model parameters determined in silico, in vitro, and with human pharmacokinetic data become progressively available. PBK modelling has been widely applied for oral/intravenous administration, but less so on topically applied chemicals. Therefore, building PBK models for topical applications and characterizing their uncertainties in the tiered approach is critical to safety decision making. The purpose of this study was to assess the confidence of PBK modelling of topically applied chemicals following the tiered framework, using non-animal methods derived parameters. Prediction of maximum plasma concentration (Cmax) and area under the curve were compared to observed kinetics from published dermal clinical studies for five chemicals (diclofenac, salicylic acid, coumarin, nicotine, caffeine). A bespoke Bayesian statistical model was developed to describe the distributions of Cmax errors between the predicted and observed data. We showed a general trend that confidence in model predictions increases when more quality in vitro data, particularly those on hepatic clearance and dermal absorption, are available as model input. The overall fold error distributions are useful for characterizing model uncertainty. We concluded that by identifying and quantifying the uncertainties in the tiered approach, we can increase the confidence in using PBK modelling to help make safety decisions on topically applied chemicals in the absence of human pharmacokinetic data.

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells.

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10µM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

A Next-Generation Risk Assessment Case Study for Coumarin in Cosmetic Products.

Next-Generation Risk Assessment is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. These principles were applied to a hypothetical safety assessment of 0.1% coumarin in face cream and body lotion. For the purpose of evaluating the use of NAMs, existing animal and human data on coumarin were excluded. Internal concentrations (plasma Cmax) were estimated using a physiologically based kinetic model for dermally applied coumarin. Systemic toxicity was assessed using a battery of in vitro NAMs to identify points of departure (PoDs) for a variety of biological effects such as receptor-mediated and immunomodulatory effects (Eurofins SafetyScreen44 and BioMap Diversity 8 Panel, respectively), and general bioactivity (ToxCast data, an in vitro cell stress panel and high-throughput transcriptomics). In addition, in silico alerts for genotoxicity were followed up with the ToxTracker tool. The PoDs from the in vitro assays were plotted against the calculated in vivo exposure to calculate a margin of safety with associated uncertainty. The predicted Cmax values for face cream and body lotion were lower than all PoDs with margin of safety higher than 100. Furthermore, coumarin was not genotoxic, did not bind to any of the 44 receptors tested and did not show any immunomodulatory effects at consumer-relevant exposures. In conclusion, this case study demonstrated the value of integrating exposure science, computational modeling and in vitro bioactivity data, to reach a safety decision without animal data.

Use of human liver and EpiSkin™ S9 subcellular fractions as a screening assays to compare the in vitro hepatic and dermal metabolism of 47 cosmetics-relevant chemicals.

The abundance of xenobiotic metabolizing enzymes (XMEs) is different in the skin and liver; therefore, it is important to differentiate between liver and skin metabolism when applying the information to safety assessment of topically applied ingredients in cosmetics. Here, we have employed EpiSkin™ S9 and human liver S9 to investigate the organ-specific metabolic stability of 47 cosmetic-relevant chemicals. The rank order of the metabolic rate of six chemicals in primary human hepatocytes and liver S9 matched relatively well. XME pathways in liver S9 were also present in EpiSkin S9; however, the rate of metabolism tended to be lower in the latter. It was possible to rank chemicals into low-, medium- and high-clearance chemicals and compare rates of metabolism across chemicals with similar structures. The determination of the half-life for 21 chemicals was affected by one or more factors such as spontaneous reaction with cofactors or non-specific binding, but these technical issues could be accounted for in most cases. There were seven chemicals that were metabolized by liver S9 but not by EpiSkin S9: 4-amino-3-nitrophenol, resorcinol, cinnamyl alcohol and 2-acetylaminofluorene (slowly metabolized); and cyclophosphamide, benzophenone, and 6-methylcoumarin. These data support the use of human liver and EpiSkin S9 as screening assays to indicate the liver and skin metabolic stability of a chemical and to allow for comparisons across structurally similar chemicals. Moreover, these data can be used to estimate the systemic bioavailability and clearance of chemicals applied topically, which will ultimately help with the safety assessment of cosmetics ingredients.

Proteomic analysis of haptenation by skin sensitisers: Diphencyprone and ethyl acrylate.

The potential risk of skin sensitisation, associated with the development of allergic contact dermatitis (ACD), is a consideration in the safety assessment of new ingredients for use in personal care products. Protein haptenation in skin by sensitising chemicals is the molecular initiating event causative of skin sensitisation. Current methods for monitoring skin sensitisation rely on limited reactivity assays, motivating interest in the development of proteomic approaches to characterise the skin haptenome. Increasing our mechanistic understanding of skin sensitisation and ACD using proteomics presents an opportunity to develop non-animal predictive methods and/or risk assessment approaches. Previously, we have used a novel stable isotope labelling approach combined with data independent mass spectrometry (HDMSE) to characterise the haptenome for a number of well-known sensitisers. We have now extended this work by characterising the haptenome of the sensitisers Diphenylcyclopropenone (DPCP) and Ethyl Acrylate (EA) with the model protein Human Serum Albumin (HSA) and the complex lysates of the skin keratinocyte, HaCaT cell line. We show that haptenation in complex nucleophilic models is not random, but a specific, low level and reproducible event. Proteomic analysis extends our understanding of sensitiser reactivity beyond simple reactivity assays and offers a route to monitoring haptenation in living cells.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

Use of a Simple in vitro Test to Assess Loss of Chemical due to Volatility during an in vitro Human Skin Absorption Study.

BACKGROUND: We tested the cutaneous distribution of 50 chemicals in frozen human skin. The mass balance (MB) values for 48% of the chemicals were < 90%, possibly due to evaporation. METHODS: We confirmed the reduction in MB was due to evaporation for two chemicals tested in skin penetration experiments using a carbon filter above the skin to trap airborne chemical. An in vitro assay was used to predict the reduction in MB due to evaporation by comparing the recovery of chemicals after 4 h of incubation at room temperature in open and closed vials. RESULTS: Evaporative losses in vitro correlated well with measured MBs (i.e., < 90%) in skin penetration experiments (R2 = 0.81). There was a correlation of the MB with the vapour pressure (VP) which could be used to group chemicals according to their likelihood to evaporate during the course of a skin penetration study. There was also a correlation of MB with Henry's law constants, melting and boiling points. CONCLUSION: Our data support the use of a quick and simple test for volatility to account for the loss of MB in skin penetration experiment due to volatility. The best parameter to indicate the potential of a chemical to evaporate is the VP.

A study of inter-individual variability in the Phase II metabolism of xenobiotics in human skin.

Understanding skin metabolism is key to improve in vitro to in vivo extrapolations used to inform risk assessments of topically applied products. However, published literature is scarce and usually covers a limited and non-representative number of donors. We developed a protocol to handle and store ex vivo skin samples post-surgery and prepare skin S9 fractions to measure the metabolic activity of Phase II enzymes. Preincubation of an excess of cofactors at 37 °C for fifteen minutes in the S9 before introduction of the testing probe, greatly increased the stability of the enzymes. Using this standardised assay, the rates of sulphation (SULT) and glucuronidation (UGT) of 7-hydroxycoumarin, methylation (COMT) of dopamine and N-acetylation (NAT) of procainamide were measured in the ng/mg protein/h (converted to ng/cm2/h) range in eighty-seven individuals. Glutathione conjugation (GST) of 1-chloro-2,4-dinitrobenzene was assessed in a smaller pool of fifty donors; the metabolic rate was much faster and measured over six minutes using a different methodology to express rates in µg/mg protein/min (converted to µg/cm2/min). A comprehensive statistical analysis of these results was carried out, separating donors by age, gender and metabolic rate measured.

A strategy for systemic toxicity assessment based on non-animal approaches: The Cosmetics Europe Long Range Science Strategy programme.

When performing safety assessment of chemicals, the evaluation of their systemic toxicity based only on non-animal approaches is a challenging objective. The Safety Evaluation Ultimately Replacing Animal Test programme (SEURAT-1) addressed this question from 2011 to 2015 and showed that further research and development of adequate tools in toxicokinetic and toxicodynamic are required for performing non-animal safety assessments. It also showed how to implement tools like thresholds of toxicological concern (TTCs) and read-across in this context. This paper shows a tiered scientific workflow and how each tier addresses the four steps of the risk assessment paradigm. Cosmetics Europe established its Long Range Science Strategy (LRSS) programme, running from 2016 to 2020, based on the outcomes of SEURAT-1 to implement this workflow. Dedicated specific projects address each step of this workflow, which is introduced here. It tackles the question of evaluating the internal dose when systemic exposure happens. The applicability of the workflow will be shown through a series of case studies, which will be published separately. Even if the LRSS puts the emphasis on safety assessment of cosmetic relevant chemicals, it remains applicable to any type of chemical.

Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome.

Skin sensitization associated with the development of allergic contact dermatitis occurs via a number of specific key events at the cellular level. The molecular initiating event (MIE), the first in the sequence of these events, occurs after exposure of the skin to an electrophilic chemical, causing the irreversible haptenation of proteins within skin. Characterization of this MIE is a key step in elucidating the skin sensitization adverse outcome pathway and is essential to providing parameters for mathematical models to predict the capacity of a chemical to cause sensitization. As a first step to addressing this challenge, we have exposed complex protein lysates from a keratinocyte cell line and human skin tissue with a range of well characterized sensitizers, including dinitrochlorobenzene, 5-chloro-2-methylisothiazol-3-one, cinnamaldehyde, and the non (or weak) sensitizer 6-methyl coumarin. Using a novel stable isotope labeling approach combined with ion mobility-assisted data independent mass spectrometry (HDMSE), we have characterized the haptenome for these sensitizers. Although a significant proportion of highly abundant proteins were haptenated, we also observed the haptenation of low abundant proteins by all 3 of the chemical sensitizers tested, indicating that within a complex protein background, protein abundance is not the sole determinant driving haptenation, highlighting a relationship to tertiary protein structure and the amino acid specificity of these chemical sensitizers and sensitizer potency.

Comparison of the Skin Penetration of 3 Metabolically Stable Chemicals Using Fresh and Frozen Human Skin.

BACKGROUND: The Cosmetics Europe ADME Task Force is developing in vitro and in silico tools for predicting skin and systemic concentrations after topical application of cosmetic ingredients. There are conflicting reports as to whether the freezing process affects the penetration of chemicals; therefore, we evaluated whether the storage of human skin used in our studies (8-12 weeks at -20°C) affected the penetration of model chemicals. METHODS: Finite doses of trans-cinnamic acid (TCA), benzoic acid (BA), and 6-methylcoumarin (6MC) (non-volatile, non-protein reactive and metabolically stable in skin) were applied to fresh and thawed frozen skin from the same donors. The amounts of chemicals in different skin compartments were analysed after 24 h. RESULTS: Although there were some statistical differences in some parameters for 1 or 2 donors, the penetration of TCA, BA, and 6MC was essentially the same in fresh and frozen skin, i.e., there were no biologically relevant differences in penetration values. Statistical differences that were evident indicated that penetration was marginally lower in frozen than in fresh skin, indicating that the barrier function of the skin was not lost. CONCLUSION: The penetration of the 3 chemicals was essentially unaffected by freezing the skin at -20°C for up to 12 weeks.

Mechanistic understanding of molecular initiating events (MIEs) using NMR spectroscopy.

Toxicological risk assessments in the 21st century are increasingly being driven by the Adverse Outcome Pathways (AOP) conceptual framework in which the Molecular Initiating Event (MIE) is of fundamental importance to pathway progression. For those MIEs that involve covalent chemical reactions, such as protein haptenation, determination of relative rates and mechanisms of reactions is a prerequisite for their understanding. The utility of NMR spectroscopy as an experimental technique for effectively providing reaction rate and mechanistic information for early assessment of likely MIE(s) has been demonstrated. To demonstrate the concept, model systems exemplifying common chemical reactions involved in the covalent modification of proteins were utilized; these involved chemical reactions of electrophilic species (representing different mechanistic classes) with simple amine and thiol nucleophiles acting as surrogates for the reactive groups of lysine and cysteine protein side chains respectively. Such molecular interactions are recognized as critical mechanisms in a variety of chemical and drug toxicities, including respiratory and skin sensitization and liver toxicity as well as being the key mechanism of action for a number of therapeutic agents.

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.

The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

Applying the skin sensitisation adverse outcome pathway (AOP) to quantitative risk assessment.

As documented in the recent OECD report 'the adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins' (OECD, 2012), the chemical and biological events driving the induction of human skin sensitisation have been investigated for many years and are now well understood. Several non-animal test methods have been developed to predict sensitiser potential by measuring the impact of chemical sensitisers on these key events (Adler et al., 2011; Maxwell et al., 2011); however our ability to use these non-animal datasets for risk assessment decision-making (i.e. to establish a safe level of human exposure for a sensitising chemical) remains limited and a more mechanistic approach to data integration is required to address this challenge. Informed by our previous efforts to model the induction of skin sensitisation (Maxwell and MacKay, 2008) we are now developing two mathematical models ('total haptenated protein' model and 'CD8(+) T cell response' model) that will be linked to provide predictions of the human CD8(+) T cell response for a defined skin exposure to a sensitising chemical. Mathematical model development is underpinned by focussed clinical or human-relevant research activities designed to inform/challenge model predictions whilst also increasing our fundamental understanding of human skin sensitisation. With this approach, we aim to quantify the relationship between the dose of sensitiser applied to the skin and the extent of the hapten-specific T cell response that would result. Furthermore, by benchmarking our mathematical model predictions against clinical datasets (e.g. human diagnostic patch test data), instead of animal test data, we propose that this approach could represent a new paradigm for mechanistic toxicology.

Application of the TTC concept to unknown substances found in analysis of foods.

Unknown substances, not previously observed, are frequently detected in foods by quality control laboratories. In many cases, the assessment of these 'new' substances requires additional chemical analysis for their identification prior to assessing risk. This identification procedure can be time-consuming, expensive and in some instances difficult. Furthermore, in many cases, no toxicological information will be available for the substance. Therefore, there is a need to develop pragmatic tools for the assessment of the potential toxicity of substances with unknown identity to avoid delays in their risk assessment. Hence, the 'ILSI Europe expert group on the application of the threshold of toxicological concern (TTC) to unexpected peaks found in food' was established to explore whether the TTC concept may enable a more pragmatic risk assessment of unknown substances that were not previously detected in food. A step-wise approach is introduced that uses expert judgement on the source of the food, information on the analytical techniques, the dietary consumption of food sources containing the unknown substance and quantitative information of the unknown substance to assess the safety to the consumer using the TTC. By following this step-wise approach, it may be possible to apply a TTC threshold of 90 µg/day for an unknown substance in food.