Review of the Evidence from Epidemiology, Toxicology, and Lung Bioavailability on the Carcinogenicity of Inhaled Iron Oxide Particulates.

Since the iron-age and throughout the industrial age, humans have been exposed to iron oxides. Here, we review the evidence from epidemiology, toxicology, and lung bioavailability as to whether iron oxides are likely to act as human lung carcinogens. Current evidence suggests that observed lung tumors in rats result from a generic particle overload effect and local inflammation that is rat-specific under the dosing conditions of intratracheal instillation. This mode of action therefore, is not relevant to human exposure. However, there are emerging differences seen in vitro, in cell uptake and cell bioavailability between "bulk" iron oxides and "nano" iron oxides. "Bulk" particulates, as defined here, are those where greater than 70% are >100 nm in diameter. Similarly, "nano" iron oxides are defined in this context as particulates where the majority, usually >95% for pure engineered forms of primary particulates (not agglomerates), fall in the range 1-100 nm in diameter. From the weight of scientific evidence, "bulk" iron oxides are not genotoxic/mutagenic. Recent evidence for "nano" iron oxide is conflicting regarding genotoxic potential, albeit genotoxicity was not observed in an in vivo acute oral dose study, and "nano" iron oxides are considered safe and are being investigated for biomedical uses; there is no specific in vivo genotoxicity study on "nano" iron oxides via inhalation. Some evidence is available that suggests, hypothetically due to the larger surface area of "nano" iron oxide particulates, that toxicity could be exerted via the generation of reactive oxygen species (ROS) in the cell. However, the potential for ROS generation as a basis for explaining rodent tumorigenicity is only apparent if free iron from intracellular "nano" scale iron oxide becomes bioavailable at significant levels inside the cell. This would not be expected from "bulk" iron oxide particulates. Furthermore, human epidemiological evidence from a number of studies suggests that iron oxide is not a human carcinogen, and therefore, based upon the complete weight of evidence, we conclude that "bulk" iron oxides are not human carcinogens.

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I).

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing.

Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing.

Non-animal approaches for consumer safety risk assessments: Unilever's scientific research programme.

Non-animal based approaches to risk assessment are now routinely used for assuring consumer safety for some endpoints (such as skin irritation) following considerable investment in developing and applying new methods over the past 20 years. Unilever's research programme into non-animal approaches for safety assessment is currently focused on the application of new technologies to risk assessments in the areas of skin allergy, cancer and general toxicity (including inhalation toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biological and chemical processes that we believe will ultimately form a sound basis for novel risk assessment approaches. Our research programme in these priority areas consists of in-house research as well as Unilever-sponsored academic research, involvement with EU-funded projects (e.g. Sens-it-iv, carcinoGENOMICS), participation in cross-industry collaborative research (e.g. COLIPA, EPAA) and ongoing involvement with other scientific initiatives on non-animal approaches to risk assessment (e.g. UK NC3Rs, US 'Human Toxicology Project' consortium).

Mass spectrometric identification of covalent adducts of the skin allergen 2,4-dinitro-1-chlorobenzene and model skin proteins.

A large proportion of allergic skin reactions are considered to be the result of skin exposure to small organic chemicals that possess the intrinsic ability to covalently modify skin proteins, either directly or following activation. In the absence of information about specific skin protein targets, studies of chemical modifications are limited to the use of model proteins. We have previously demonstrated that selected well known skin sensitizers (2,4-dinitro-1-chlorobenzene and phenyl salicylate) have the ability to covalently modify residues selectively on the model protein, human serum albumin. In the present work, we focus on the differences in covalent binding observed for two additional model proteins, human cytokeratin 14 and human cofilin, both constituent proteins of skin. Using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nano LC-MS and -MS/MS strategies, the amino acid residues targeted by 2,4-dinitro-1-chlorobenzene on the two model proteins have been identified. In contrast, a structurally related non-sensitiser (2,4-dichloro-1-nitrobenzene) and a non-sensitising irritant (benzalkonium chloride) did not covalently modify the model proteins. Detailed examination of the results for the sensitizers indicate that reactive chemicals target nucleophilic amino acids residing in specific microenvironments of the 3D protein structure that are conducive to reactivity. This observation has important implications for the development of hapten-peptide binding assays. It is envisaged that the data from such assays will be integrated with outputs from other in vitro assays in the future to give a prediction of the sensitisation potential of novel chemicals.

Assuring consumer safety without animal testing: a feasibility case study for skin sensitisation.

Allergic Contact Dermatitis (ACD; chemical-induced skin sensitisation) represents a key consumer safety endpoint for the cosmetics industry. At present, animal tests (predominantly the mouse Local Lymph Node Assay) are used to generate skin sensitisation hazard data for use in consumer safety risk assessments. An animal testing ban on chemicals to be used in cosmetics will come into effect in the European Union (EU) from March 2009. This animal testing ban is also linked to an EU marketing ban on products containing any ingredients that have been subsequently tested in animals, from March 2009 or March 2013, depending on the toxicological endpoint of concern. Consequently, the testing of cosmetic ingredients in animals for their potential to induce skin sensitisation will be subject to an EU marketing ban, from March 2013 onwards. Our conceptual framework and strategy to deliver a non-animal approach to consumer safety risk assessment can be summarised as an evaluation of new technologies (e.g. 'omics', informatics), leading to the development of new non-animal (in silico and in vitro) predictive models for the generation and interpretation of new forms of hazard characterisation data, followed by the development of new risk assessment approaches to integrate these new forms of data and information in the context of human exposure. Following the principles of the conceptual framework, we have been investigating existing and developing new technologies, models and approaches, in order to explore the feasibility of delivering consumer safety risk assessment decisions in the absence of new animal data. We present here our progress in implementing this conceptual framework, with the skin sensitisation endpoint used as a case study.

Development of a modified in vitro skin absorption method to study the epidermal/dermal disposition of a contact allergen in human skin.

In vitro skin absorption methods exist in Organisation for Economic Co-operation and Development (OECD) guideline form (No. 428) and are used to estimate the degree of systemic penetration of chemicals through skin. More detailed kinetics of permeation through skin compartments are not described well by existing methods. This study was designed to assess the practical feasibility of generating compartmental (stratum corneum/epidermal/dermal) disposition and kinetic data of topically applied chemicals. For chemically induced effects initiated in the skin (e.g., skin allergy), the delivery of tissue concentrations of chemical will impact the incidence and severity of biological effect. Explicit data on the kinetics of chemical disposition in skin have not traditionally been needed for skin allergy risk assessment: current in vivo assays embody delivery implicitly. Under the 7th Amendment to the European Cosmetics Directive, in vivo assays (such as the local lymph node assay for skin sensitization) will not be permitted to assess cosmetic ingredients. New in vitro and in silico alternative approaches and ways of predicting risk of adverse effects in humans need to be developed, and new methods such as that described here provide a way of estimating delivered concentrations and the effect of formulation changes on that delivery. As we continue to deconstruct the contributing factors of skin allergy in humans, it will be useful to have methods available that can measure skin tissue compartment exposure levels delivered from different exposure use scenarios. Here we provide such a method. The method could also be used to generate useful data for developing in silico kinetic models of compartmental skin delivery and for refining data for skin delivery in relation to the evaluation of systemic toxicity.

Xenobiotic metabolism in human skin and 3D human skin reconstructs: a review.

In this review, we discuss and compare studies of xenobiotic metabolism in both human skin and 3D human skin reconstructs. In comparison to the liver, the skin is a less studied organ in terms of characterising metabolic capability. While the skin forms the major protective barrier to environmental chemical exposure, it is also a potential target organ for adverse health effects. Occupational, accidental or intended-use exposure to toxic chemicals could result in acute or delayed injury to the skin (e.g. inflammation, allergy, cancer). Skin metabolism may play a role in the manifestation or amelioration of adverse effects via the topical route. Today, we have robust testing strategies to assess the potential for local skin toxicity of chemical exposure. Such methods (e.g. the local lymph node assay for assessing skin sensitisation; skin painting carcinogenicity studies) incorporate skin metabolism implicitly in the in vivo model system used. In light of recent European legislation (i.e. 7(th) Amendment to the Cosmetics Directive and Registration Evaluation and Authorisation of existing Chemicals (REACH)), non-animal approaches will be required to reduce and replace animal experiments for chemical risk assessment. It is expected that new models and approaches will need to account for skin metabolism explicitly, as the mechanisms of adverse effects in the skin are deconvoluted. 3D skin models have been proposed as a tool to use in new in vitro alternative approaches. In order to be able to use 3D skin models in this context, we need to understand their metabolic competency in relation to xenobiotic biotransformation and whether functional activity is representative of that seen in human skin.

Investigating protein haptenation mechanisms of skin sensitisers using human serum albumin as a model protein.

Covalent modification of skin proteins by electrophiles is a key event in the induction of skin sensitisation but not skin irritation although the exact nature of the binding mechanisms has not been determined empirically for the vast majority of sensitisers. It is also unknown whether immunologically relevant protein targets exist in the skin contributing to effecting skin sensitisation. To determine the haptenation mechanism(s) and spectra of amino acid reactivity in an intact protein for two sensitisers expected to react by different mechanisms, human serum albumin (HSA) was chosen as a model protein. The aim of this work was also to verify for selected non-sensitisers and irritants that no protein haptenation occurs even under forcing conditions. HSA was incubated with chemicals and the resulting complexes were digested with trypsin and analysed deploying matrix-assisted laser desorption/ionization mass spectrometry, reverse phase high performance liquid chromatography and nano-electrospray tandem mass spectrometry. The data confirmed that different residues (lysine, cysteine, histidine and tyrosine) are covalently modified in a highly selective and differential manner by the sensitisers 2,4-dinitro-1-chlorobenzene and phenyl salicylate. Additionally, non-sensitisers 2,4-dichloro-1-nitrobenzene, butyl paraben and benzaldehyde and irritants benzalkonium chloride and sodium dodecyl sulphate did not covalently modify HSA under any conditions. The data indicate that covalent haptenation is a prerequisite of skin sensitisation but not irritation. The data also suggest that protein modifications are targeted to certain amino acids residing in chemical microenvironments conducive to reactivity within an intact protein. Deriving such information is relevant to our understanding of antigen formation in the immunobiology of skin sensitisation and in the development of in vitro protein haptenation assays.

Systematic review in chemical risk assessment - A chemical industry perspective.

This commentary provides a perspective from the chemicals industry on the potential usefulness of systematic review approaches in chemical risk assessment.

Implementing systematic review techniques in chemical risk assessment: Challenges, opportunities and recommendations.

Systematic review (SR) is a rigorous, protocol-driven approach designed to minimise error and bias when summarising the body of research evidence relevant to a specific scientific question. Taking as a comparator the use of SR in synthesising research in healthcare, we argue that SR methods could also pave the way for a "step change" in the transparency, objectivity and communication of chemical risk assessments (CRA) in Europe and elsewhere. We suggest that current controversies around the safety of certain chemicals are partly due to limitations in current CRA procedures which have contributed to ambiguity about the health risks posed by these substances. We present an overview of how SR methods can be applied to the assessment of risks from chemicals, and indicate how challenges in adapting SR methods from healthcare research to the CRA context might be overcome. Regarding the latter, we report the outcomes from a workshop exploring how to increase uptake of SR methods, attended by experts representing a wide range of fields related to chemical toxicology, risk analysis and SR. Priorities which were identified include: the conduct of CRA-focused prototype SRs; the development of a recognised standard of reporting and conduct for SRs in toxicology and CRA; and establishing a network to facilitate research, communication and training in SR methods. We see this paper as a milestone in the creation of a research climate that fosters communication between experts in CRA and SR and facilitates wider uptake of SR methods into CRA.

Cinnamic compound metabolism in human skin and the role metabolism may play in determining relative sensitisation potency.

BACKGROUND: trans-Cinnamaldehyde and trans-cinnamic alcohol cause allergic contact dermatitis (ACD) in humans; cinnamaldehyde is a more potent sensitiser than cinnamic alcohol. These two chemicals are principal constituents of the European Standard 'Fragrance Mix', as used in patch testing diagnostics of sensitisation to fragrances by clinical dermatologists. As contact sensitisers are usually protein reactive compounds, it is hypothesised that cinnamic alcohol (not protein-reactive) is a 'prohapten' that requires metabolic activation, presumably by cutaneous oxidoreductases, to the protein-reactive cinnamaldehyde (a 'hapten'). It is postulated that cinnamaldehyde can be detoxified by aldehyde dehydrogenase (ALDH) to cinnamic acid and/or by alcohol dehydrogenase (ADH) to cinnamic alcohol. Hence, a variety of metabolic pathways may contribute to the relative exposures and hence sensitising potencies of cinnamic alcohol and cinnamaldehyde. OBJECTIVE: To evaluate the extent of cinnamaldehyde and cinnamic alcohol metabolism in human skin and provide evidence for the role of cutaneous ADH and ALDH in such metabolism. METHODS: The extent of cinnamic alcohol and aldehyde metabolism was investigated in human skin homogenates and sub-cellular fractions. A high performance liquid chromatography method was used for analysis of skin sample extracts. Studies were conducted in the presence and absence of the ADH/cytochrome P450 inhibitor 4-methylpyrazole and the cytosolic ALDH inhibitor, disulfiram. RESULTS: Differential metabolism of cinnamic alcohol and cinnamaldehyde was observed in various subcellular fractions: skin cytosol was seen to be the major site of cinnamic compound metabolism. Significant metabolic inhibition was observed using 4-methylpyrazole and disulfiram in whole skin homogenates and cytosolic fractions only. CONCLUSIONS: This study has demonstrated that cutaneous ADH and ALDH activities, located within defined subcellular compartments, play important roles in the activation and detoxification of CAlc and CAld in skin. Such findings are important to the development of computational hazard prediction tools for sensitisation (e.g. the DEREK program) and also to dermatologists in understanding observed interindividual differences, cross-reactivities or co-sensitisation to different cinnamic compounds in the clinic.

From xenobiotic chemistry and metabolism to better prediction and risk assessment of skin allergy.

Allergic contact dermatitis (ACD) is a condition that can have a serious impact on quality of life. The manifestation of ACD is dependent upon the primary sensitisation of an individual to a specific substance following skin exposure. It is important to identify and manage the risks associated with exposure to known skin sensitisers, in both the manufacture and use of consumer products. At present, the only validated approaches to conclusively identify sensitisation hazard and estimate potency are in vivo models such as the local lymph node assay. No in vitro test methods exist for this endpoint. There is an urgent need to develop novel in vitro/in silico testing or risk assessment strategies to replace animal testing. It is envisaged that such novel approaches can only be developed on the foundation of a good mechanistic understanding of skin sensitisation. Early stages of sensitisation are thought to be dependent upon the extent of compound absorption and bioavailability, rates of metabolic activation or detoxification and intrinsic reactivity of the bioavailable xenobiotic electrophile with skin protein nucleophiles. This review explores general chemical and metabolic aspects in relation to the potential formation of protein-hapten conjugates. Despite the complexities and poor understanding of some of the metabolic processes involved in skin sensitisation, it is possible to describe some of the relationships between chemical structures and the ability to form covalent conjugates with proteins. A prototypical group of xenobiotics that have been used to explore sensitisation mechanisms in some detail are selected cinnamic derivatives: a discussion of recent work using these compounds is presented as a case study. Novel aspects for future research in this area are also discussed.

Species variations in cutaneous alcohol dehydrogenases and aldehyde dehydrogenases may impact on toxicological assessments of alcohols and aldehydes.

Alcohol dehydrogenase (ADH; EC. 1.1.1.1) and aldehyde dehydrogenase (ALDH; EC 1.2.1.3) play important roles in the metabolism of both endogenous and exogenous alcohols and aldehydes. The expression and localisation patterns of ADH (1-3) and ALDH (1-3) were investigated in the skin and liver of the mouse (BALB/c and CBA/ca), rat (F344) and guinea-pig (Dunkin-Hartley), using Western blot analysis and immunohistochemistry with class-specific antisera. ALDH2 expression and localisation was also determined in human skin, while ethanol oxidation, catalysed by ADH, was investigated in the mouse, guinea-pig and human skin cytosol. Western blot analysis revealed that ADH1, ADH3, ALDH1 and ALDH2 were expressed, constitutively, in the skin and liver of the mouse, rat and guinea-pig. ADH2 was not detected in the skin of any rodent species/strain, but was present in all rodent livers. ALDH3 was expressed, constitutively, in the skin of both strains of mouse and rat, but was not detected in guinea-pig skin and was absent in all livers. Immunohistochemistry showed similar patterns of expression for ADH and ALDH in both strains of mouse, rat, guinea-pig and human skin sections, with localisation predominantly in the epidermis, sebaceous glands and hair follicles. ADH activity (apparent V(max), nmoles/mg protein/min) was higher in liver (6.02-16.67) compared to skin (0.32-1.21) and lower in human skin (0.32-0.41) compared to mouse skin (1.07-1.21). The ADH inhibitor 4-methyl pyrazole (4-MP) reduced ethanol oxidation in the skin and liver in a concentration dependent manner: activity was reduced to approximately 30-40% and approximately 2-10% of the control activity, in the skin and liver, respectively, using 1 mM 4-MP. The class-specific expression of ADH and ALDH enzymes, in the skin and liver and their variation between species, may have toxicological significance, with respect to the metabolism of endogenous and xenobiotic alcohols and aldehydes.

Determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment.

Development of risk assessment methods for skin sensitization in the absence of toxicological data generated in animals represents a major scientific and technical challenge. The first step in human skin sensitization induction is the transport of sensitizer from the applied dose on the skin surface to the epidermis, where innate immune activation occurs. Building on the previous development of a time course in vitro human skin permeation assay, new kinetic data for 10 sensitizers and 2 nonsensitizers are reported. Multicompartmental modeling has been applied to analyze the data and determine candidate dose parameters for use in integrated risk assessment methods: the area under the curve (AUC) and maximum concentration (C(max)) in the epidermis. A model with two skin compartments, representing the stratum corneum and viable skin (epidermis and dermis), was chosen following a formal model selection process. Estimates of the uncertainty, as well as average values of the epidermal disposition kinetics parameters, were made by fitting to the time course skin permeation data from individual skin donors. A potential reduced time course method is proposed based on two time points at 4 and 24 h, which gives results close to those from the full time course for the current data sets. The time course data presented in this work have been provided as a resource for development of predictive in silico skin permeation models.

Assuring safety without animal testing: Unilever's ongoing research programme to deliver novel ways to assure consumer safety.

Assuring consumer safety without the generation of new animal data is currently a considerable challenge. However, through the application of new technologies and the further development of risk-based approaches for safety assessment, we remain confident it is ultimately achievable. For many complex, multi-organ consumer safety endpoints, the development, evaluation and application of new, non-animal approaches is hampered by a lack of biological understanding of the underlying mechanistic processes involved. The enormity of this scientific challenge should not be underestimated. To tackle this challenge a substantial research programme was initiated by Unilever in 2004 to critically evaluate the feasibility of a new conceptual approach based upon the following key components: 1.Developing new, exposure-driven risk assessment approaches. 2.Developing new biological (in vitro) and computer-based (in silico) predictive models. 3.Evaluating the applicability of new technologies for generating data (e.g. "omics", informatics) and for integrating new types of data (e.g. systems approaches) for risk-based safety assessment. Our research efforts are focussed in the priority areas of skin allergy, cancer and general toxicity (including inhaled toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biology and molecular mechanisms that we believe will ultimately form a sound basis for novel risk assessment approaches. Our research programme in these priority areas consists of in-house research as well as Unilever-sponsored academic research, involvement in EU-funded projects (e.g. Sens-it-iv, Carcinogenomics), participation in cross-industry collaborative research (e.g. Colipa, EPAA) and ongoing involvement with other scientific initiatives on non-animal approaches to risk assessment (e.g. UK NC3Rs, US "Human Toxicology Project" consortium).

Measurement of CD4+ and CD8+ T-lymphocyte cytokine secretion and gene expression changes in p-phenylenediamine allergic patients and tolerant individuals.

Factors predisposing to individual susceptibility to contact allergic dermatitis are ill defined. This study was designed to characterize the response of allergic and tolerant individuals' T-lymphocytes after exposure to p-phenylenediamine (PPD). Peripheral blood mononuclear cells (PBMCs) from allergic patients proliferated when treated with PPD and Bandrowski's base (BB) and secreted IL-1alpha, -1beta, -4, -5, -6, -8, -10, and -13; IFN-gamma; tumor necrosis factor-alpha; MIP-1alpha/beta; MCP-1 (monocyte chemotactic protein-1); and RANTES. PBMCs from tolerant individuals were stimulated to proliferate only with BB, and they secreted significantly lower levels of Th2 cytokines. Principal component analysis showed that genes are differentially expressed between the patient groups. A network-based analysis of microarray data showed upregulation of T helper type 2 (Th2) gene pathways, including IL-9, in allergic patients, but a regulatory gene profile in tolerant individuals. Real-time PCR confirmed the observed increase in Th2 cytokine gene transcription in allergic patients. Purified CD4+ and CD8+ T cells from allergic patients were stimulated to proliferate and secrete Th2 cytokines following antigen exposure. Only CD4+ T cells from tolerant individuals were stimulated by BB, and levels of Th2 cytokines were 80% lower. The nature of the antigenic determinant stimulating PBMCs and levels of Th2 cytokines, including IL-9, was confirmed in a validation cohort. These studies show increased activity of Th2 cytokines in CD4+ and CD8+ T cells from individuals with allergic contact dermatitis.