A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

Safety assessment of a traditionally used extract from leaves of Boldoa purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: Boldoa purpurascens Cav. (Nyctaginaceae) is a plant species used in traditional medicine in Cuba as a diuretic. AIM OF THE STUDY: The aim of the present investigation was to evaluate the safety profile of a hydroalcoholic extract from leaves of Boldoa purpurascens. MATERIALS AND METHODS: First, an experimental study to assess the oral acute toxicity at a dose of 2000mg/kg body weight of the extract was carried out. Potential genotoxicity of the extract was evaluated using the Ames test and the micronucleus induction assay in mouse bone marrow. In the Ames test a concentration range of 50, 100, 150, 300 and 500µg/plate was tested. In the micronucleus induction assay, doses of 500, 1000 and 2000mg/kg of body weight were tested. For completeness, since the extract contains saponins, the evaluation of the hemolytic activity, ocular and skin irritation were included. RESULTS: No signs or symptoms of toxicity were observed in the oral acute toxicity test (body weight at baseline, seven days and end of the experiment of 236.41±20.07, 256.81±30.44 and 240.02±26.16 respectively for the treated group). The hydroalcoholic extract from the leaves was not mutagenic in the Ames test, and no genotoxicity was observed in the micronucleus assay. A hemolysis test at concentration of 1mg/mL confirmed hemolytic activity, which is not a safety concern since saponins are not absorbed after oral administration. In order to evaluate the percentage of protein denaturation, the ocular irritability index was calculated. The extract was found to be irritating. Finally, skin irritability was evaluated and the irritation index was equal to zero. CONCLUSIONS: Based on the toxicological evaluation of a traditionally used hydroalcoholic extract from the leaves of Boldoa purpurascens we can confirm the safety of its oral use.

Compensation for occupational skin diseases.

The Korean list of occupational skin diseases was amended in July 2013. The past list was constructed according to the causative agent and the target organ, and the items of that list had not been reviewed for a long period. The revised list was reconstructed to include diseases classified by the International Classification of Diseases (10th version). Therefore, the items of compensable occupational skin diseases in the amended list in Korea comprise contact dermatitis; chemical burns; Stevens-Johnson syndrome; tar-related skin diseases; infectious skin diseases; skin injury-induced cellulitis; and skin conditions resulting from physical factors such as heat, cold, sun exposure, and ionized radiation. This list will be more practical and convenient for physicians and workers because it follows a disease-based approach. The revised list is in accordance with the International Labor Organization list and is refined according to Korean worker's compensation and the actual occurrence of occupational skin diseases. However, this revised list does not perfectly reflect the actual status of skin diseases because of the few cases of occupational skin diseases, incomplete statistics of skin diseases, and insufficient scientific evidence. Thus, the list of occupational diseases should be modified periodically on the basis of recent evidence and statistics.

Reactivity profiling: covalent modification of single nucleophile peptides for skin sensitization risk assessment.

The molecular basis of chemical allergy is rooted in the ability of an allergen (hapten) to modify endogenous proteins. This mechanistic understanding aided development of screening assays which generate reproducible quantitative and qualitative reactivity data. Such assays use model peptides with a limited number and type of protein nucleophiles, and the data does not reflect the specificity, variety, and complexity of hapten interactions with multiple nucleophiles. Building on these developments, we extended the standardized approach to maximize the type and the amount of information that can be derived from an in chemico assay. We used a panel of six single nucleophile peptides and individually optimized the incubation conditions to favor chemical modification. Employing liquid chromatography tandem mass spectrometry (LC-MS/MS) technique, we simultaneously obtained multiple quantitative and qualitative measurements (% peptide depletion, adducts formation, and peptide dimerization for Cys-containing peptide). Using these methods, we obtained reactivity data for 36 chemicals of known skin sensitizing potency. By optimizing incubation conditions, we ensured detection of all reactive chemicals. We explored the LC-MS/MS approach to generate kinetic data for 10 chemicals allowing further characterization of reactivity and a potentially more robust quantitative reactivity descriptor. Our ultimate aim is to integrate this dataset with available physicochemical data and outputs from other predictive assays, all addressing different key steps in the induction of sensitization, to help us make decisions about the safe use of chemicals without using animal tests. The epidermal protein target sites, modification of which may be immunogenic and lead to induction of skin sensitization, are currently unknown. Increasing the understanding of this process may help further refine in chemico reactivity assays as well as aid the interpretation of the reactivity data.

Skin sensitization in chemical risk assessment: report of a WHO/IPCS international workshop focusing on dose-response assessment.

An international workshop was held in 2006 to evaluate experimental techniques for hazard identification and hazard characterization of sensitizing agents in terms of their ability to produce data, including dose-response information, to inform risk assessment. Human testing to identify skin sensitizers is discouraged for ethical reasons. Animal-free alternatives, such as quantitative structure-activity relationships and in vitro testing approaches, have not been sufficiently developed for such application. Guinea pig tests do not generally include dose-response assessment and are therefore not designed for the assessment of potency, defined as the relative ability of a chemical to induce sensitization in a previously naive individual. In contrast, the mouse local lymph node assay does include dose-response assessment and is appropriate for this purpose. Epidemiological evidence can be used only under certain circumstances for the evaluation of the sensitizing potency of chemicals, as it reflects degree of exposure as well as intrinsic potency. Nevertheless, human diagnostic patch test data and quantitative elicitation data have provided very important information in reducing allergic contact dermatitis risk and sensitization in the general population. It is therefore recommended that clinical data, particularly dose-response data derived from sensitized patients, be included in risk assessment.

Assessment of the U937 cell line for the detection of contact allergens.

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1beta and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times. Test item-specific modulations of the chosen activation markers (CD86, IL-1beta and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.

Histological examination in assessment of ultraviolet-induced suppression of contact hypersensitivity response.

The evaluation of contact hypersensitivity (CHS) reaction is one of the methods used in the assessment of the immune status of an organism after UV radiation. The aim of the study was to compare usefulness of visual scoring system and histological morphometry in the assessment of CHS response after exposure of humans to solar simulated radiation (SSR). The study included 140 healthy volunteers, 33 people were irradiated for 2 days, 34 - for 10 days and 33 - for 30 days with SSR. Forty non-irradiated individuals served as controls. All the volunteers were sensitized with diphenylocyclopropenone (DPCP) 24 h after final exposure. Statistical analysis comparing intensity of CHS reaction based on visual score between irradiated groups and non-irradiated group revealed no differences (p>0.05). We found a significant difference in epidermal thickness between healthy skin and irradiated groups (p<0.05) and a positive correlation between intensity of spongiosis and clinical score for CHS response at 3.2 DPCP site (p<0.000001). A negative correlation between time of irradiation and spongiosis score was revealed (R=-0.28; p<0.001). We conclude that histological examination of biopsies taken from one of the series of elicitation sites is a reliable and sensitive method in the evaluation of CHS response after UVR.

A farmer's occupational airborne contact dermatitis masqueraded by coexisting rosacea: delayed diagnosis and legal acknowledgement.

A rare case of coexistence of occupational airborne dermatitis with rosacea is presented in a 41-year-old female farmer. Her first dermatitis symptoms appeared at the age of 10 when she started helping her parents on the farm. Uncovered skin areas of the face, neck, décolleté, forearms and the hands gradually became involved. The dermatitis symptoms were provoked by agricultural dusts (especially of flax and dried herbs). For the subsequent 30 years, the work-related disease remained undiagnosed due to the lack of pre-employment and periodical health check in agriculture. She also suffered from protein contact dermatitis of the hands from cow epithelium. About 20 years after the onset of airborne dermatitis, rosacea developed, possibly secondary to the prolonged treatment. Diagnostic tests carried out at our department confirmed hypersensitivity to occupational allergens: type I allergy to storage mites, moulds, and cow epithelium. A cutaneous late-phase reaction on prick tests and serum precipitins to the bacterium Pantoea agglomerans (Erwinia herbicola) also were found. Among non-occupational hypersensitivities, type I allergy to house dust mites and contact allergy to methylchloroisothiazolinone/methylisothiazolinone (Kathon CG) was found. In connection with these results, the significance of agricultural dusts in farmers' airborne dermatitis is discussed. Also presented are the problems with obtaining acceptance from the State Sanitary Authority for qualification of this case as an occupational disease, which was due to the coexistence of the non-occupational rosacea. Discussed is also the problem of pre-employment exposure to occupational allergens among farmers' children, and the difficulties with delivering occupational health services to self-employed farmers.

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.

Assessment of glycosylation-dependent cell adhesion molecule 1 as a correlate of allergen-stimulated lymph node activation.

Early changes in gene expression have been identified by cDNA microarray technology. Analysis of draining auricular lymph node tissue sampled at 48 h following exposure to the potent contact allergen 2,4-dinitrofluorobenzene (DNFB) provided examples of up- and down-regulated genes, including onzin and guanylate binding protein 2, and glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), respectively. Allergen-induced changes in these three genes were confirmed in dose-response and kinetic analyses using Northern blotting and/or reverse transcription-polymerase chain reaction techniques. The results confirmed that these genes are robust and relatively sensitive markers of early changes provoked in the lymph node by contact allergen. Upon further investigation, it was found that altered expression of the adhesion molecule GlyCAM-1 was not restricted to treatment with DNFB. Topical sensitization of mice to a chemically unrelated contact allergen, oxazolone, was also associated with a decrease in the expression of mRNA for GlyCAM-1. Supplementary experiments revealed that changes in expression of this gene are independent of the stimulation by chemical allergens of proliferative responses by draining lymph node cells. Taken together these data indicate that the expression of GlyCAM-1 is down-regulated rapidly following epicutaneous treatment of mice with chemical allergens, but that this reduction is associated primarily with changes in lymph node cell number, or some other aspect of lymph node activation, rather than proliferation.

What's your assessment?

The "What's Your Assessment?" series includes a short case presentation and differential diagnosis. It is followed by a discussion of the disease or condition and the rationale used in each step of the assessment.

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy.

Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered.

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

Examination of the local lymph node assay for use in contact sensitization risk assessment.

The purpose of this study was to evaluate the utility of the murine local lymph node assay (LLNA) for contact sensitization risk assessment. Cellular proliferative activity in draining lymph nodes was determined for individual animals on Day 5 following four daily epicutaneous applications of the test chemical to the ears. Seventeen chemicals were tested, covering a range of materials including preservatives, drug actives, and perfume raw materials. The assay was found to be useful for identifying strong, moderate, and some weak sensitizers as defined by other testing methods (guinea pig, human). For evaluating the antigen specificity of the LLNA proliferative response, an in vitro blastogenesis assay was used. Dendritic cells (DC) isolated from lymph nodes of mice treated 24 hr earlier with trinitrochlorobenzene (TNCB) were capable of in vitro stimulation of lymphocytes from TNCB-sensitized mice, but not lymphocytes from mice sensitized to the preservative mixture of 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI). Conversely, DC from mice treated 24 hr earlier with MCI/MI were capable of stimulating lymphocytes from MCI/MI-sensitized mice, but were unable to stimulate lymphocytes from TNCB-sensitized mice, demonstrating the specificity of the response. The results of these studies support the use of the murine LLNA for both investigative and predictive contact sensitization testing. The LLNA offers the advantages of requiring less time for completion, incorporating an objective endpoint, requiring approximately half the number of animals, and being less costly than most currently employed guinea pig test methods. In addition, we believe the murine LLNA is a useful test to incorporate into a scheme for contact sensitization risk assessment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal model for assessment of skin irritancy.

Irritant skin reactions from repeated open applications of low concentrations of sodium lauryl sulphate (SLS) have been studied macroscopically and microscopically in guinea pigs. After 3 applications daily for 3 days, 2% SLS aqueous solutions gave a naked eye assessment, increase in epidermal thickness and total dermal inflammatory cell response, which was greater than for a 1% SLS solution. The dermal inflammatory cell response was mainly mononuclear (lymphocytic) in nature. With the SLS reactions as control, various organic solvents were studied and ranked against the SLS reactions and internally. Trichlorethylene was the most irritant solvent, ranking as high as 2% SLS. Other chlorinated hydrocarbons and aromatic hydrocarbons tested caused irritant reactions. The alcohols and acetone gave no reaction. White spirit was as irritant as trichlorethylene. Thinners were less irritant, around the level of the 1% SLS control reaction. The 4-day experimental design is a convenient and suitable animal model for screening irritant potential, and provides information relevant to the pathogenesis of irritant contact reactions.

Topical effects of molten salt on rat integument: a histological and photometric assessment.

The topical effects of the molten salt 1-methyl-3-ethylimidazolium tetrachloroaluminate (melt) and its organic component, the organic salt 1-methyl-3-ethylimidazolium chloride were tested on rat integument. Evaluation was accomplished using standard histological techniques supplemented with digital analysis using the microscope photometer. Two groups of animals were treated with 1.5 ml of either the melt or the organic salt for 10 consecutive days. A third group treated with the melt had the treated area flushed with running water 5 min after each application (wash). Significant treatment effects were observed in rats treated with the melt and wash preparations while the effects of the organic salt were unremarkable. The melt induced an ulcerative dermatitis with acanthosis while the wash produced only mild acanthosis and dermatitis. This damage appears to result from the penetration of aluminum chloride in the melt through the skin and its toxic effects on the cells of the dermis and epidermis.