Richard Cubberley

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.

Comparison of protocols for measuring cosmetic ingredient distribution in human and pig skin

The Cosmetics Europe Skin Bioavailability and Metabolism Task Force aims to improve the measurement and prediction of the bioavailability of topically-exposed compounds for risk assessment. Key parameters of the experimental design of the skin penetration studies were compared. Penetration studies with frozen human and pig skin were conducted in two laboratories, according to the SCCS and OECD 428 guidelines. The disposition in skin was measured 24h after finite topical doses of caffeine, resorcinol and 7-ethoxycoumarin. The bioavailability distribution in skin layers of cold and radiolabelled chemicals were comparable. Furthermore, the distribution of each chemical was comparable in human and pig skin. The protocol was reproducible across the two laboratories. There were small differences in the amount of chemical detected in the skin layers, which were attributed to differences in washing procedures and anatomical sites of the skin used. In conclusion, these studies support the use of pig skin as an alternative source of skin should the availability of human skin become a limiting factor. If radiolabelled chemicals are not available, cold chemicals can be used, provided that the influence of chemical stability, reactivity or metabolism on the experimental design and the relevance of the data obtained is considered.

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.

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.

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 37u202f°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.