Identification of potential biomarkers for predicting acute dermal irritation by proteomic analysis

In vitro alternative tests aiming at replacing the traditional animal test for predicting the irritant potential of chemicals have been developed, but the assessment parameters or endpoints are still not sufficient for analysis. To discover novel endpoints for skin irritation responses, a proteomics approach was used to analyze the protein expression in human keratinocytes exposed to sodium lauryl sulfate in the present study. Among the 20 identified proteins with altered expression, small heat shock protein 27 (HSP27) and superoxide dismutase [Cu-Zn] were down-regulated while cofilin-1 was up-regulated significantly in response to the chemical challenge. Keratinocytes were exposed to acid and basic chemicals for further validation of the proteins. HSP27 displayed the most significant alteration both in mRNA and protein levels, accompanied by nuclear translocation. The irritation also induced an increased production of interleukin-1α in keratinocytes. These findings suggest that these proteins may be combinational biomarkers or additional endpoints for skin hazard assessment. Further investigation into the protein alterations would be helpful for the mechanistic understanding of skin irritation.     

Heavy metals chromium and neodymium reduced phosphorylation level of heat shock protein 27 in human keratinocytes

Heavy metals may exert their acute and chronic effects on the human skin through stress signals. In the present study, 2DE-based proteomics was used to analyze the protein expression in human keratinocytes exposed to heavy metals, chromium and neodymium, and 10 proteins with altered expression were identified. Among these proteins, small heat shock protein 27 (HSP27) was up-regulated significantly and the up-regulation was validated by Western blot and immunofluorescence. In addition, the mRNA expression level of HSP27 markedly increased as detected by quantitative PCR. More interestingly, the ratio of phosphorylated HSP27 and total HSP27 significantly decreased in keratinocytes treated with the heavy metals. These findings suggested that heavy metals reduced the phosphorylation level of HSP27, and that the ratio of p-HSP27 and HSP27 may represent a potential marker or additional endpoint for the hazard assessment of skin irritation caused by chemical products.     

The usefulness of toxicogenomics for predicting acute skin irritation on in vitro reconstructed human epidermis

In vitro models aiming at replacing the traditional animal test for determining the skin irritation potential of a test substance have been developed, evaluated in prevalidation studies and recently validated by the European Center for the Validation of Alternative Methods (ECVAM). To investigate the usefulness of toxicogenomic technologies to identify novel mechanistic endpoints for skin irritation responses, the present work challenged the human reconstituted epidermis model validated by ECVAM with four irritant chemicals and four non-classified chemicals tested at subcytotoxic concentrations. Using a specifically designed low-density DNA array, about 50 genes out of 240 were found to be significantly and differentially expressed between tissues exposed to irritant and non-irritant chemicals for at least one test chemical when compared to the seven others. These genes are involved in cell signalling, stress response, cell cycle, protein metabolism and cell structure. Among them, 16 are expressed in the same way whatever the irritant compound applied. The differential gene expressions might represent new or additional endpoints useful for the mechanistic understanding and perhaps also the hazard assessment of the skin irritation potential of chemicals and products.     

An in vitro model for detecting skin irritants: methyl green-pyronine staining of human skin explant cultures.

We evaluated the potential of human organotypic skin explant cultures (hOSECs) for screening skin irritants. Test chemicals were applied to the epidermis of the skin explants which were incubated for 4, 24 or 48 h in tissue culture medium. A decrease in epidermal RNA staining, visualised in frozen sections using a modified methyl-green pyronine (MGP) staining procedure, was used as a marker of irritancy. A decrease in epidermal RNA after a 4-, 24- or 48-h exposure to a certain concentration of a test chemical equated to a MGP score of 3, 2 or 1, respectively. The MGP score was 0 if there was no keratinocyte cytotoxicity after a 48-h exposure. A minimum of three donors were used per chemical and the average MGP score was used to classify the chemical as irritant or not. Chemicals with an average MGP score > or =1.5 were classified as irritants (R38), at that concentration. Chemicals with a MGP score <1.5 were not classified (NC), at that concentration. The results obtained using human skin in vitro were compared with published data obtained using cultured porcine skin, the cutaneous Draize test (from this point referred to as the "rabbit skin irritation test") and volunteer studies. There was an excellent correlation between the classification of a chemical, as R38 or NC, based on hOSEC and results of volunteer studies. The hOSEC model predicted perfectly the irritation hazard of the 22 chemicals for which volunteer data were available. The porcine OSEC correctly predicted the classification of 21 of 22 (95%) chemicals and the rabbit skin irritation test correctly predicted the classification of 14 of 15 chemicals (93%) for which data were available. In conclusion, MGP staining of human skin explant cultures can be used to predicted human skin irritancy in vivo. In addition, the data validate the use of porcine skin as an alternative to human skin for screening for dermal irritants in vitro.     

Development of an in vitro corrosion/irritation prediction assay using the EpiDerm skin model.

A validation of the in vitro skin corrosion method using the EpiDerm skin model was performed using 12 recommended chemicals. All chemicals were correctly classified by OECD test guideline 431. In order to predict corrosion and/or irritation potential, additional compound exposure times and IL-1alpha measurements were included in a tiered testing approach. Four exposure times were performed followed by MTT (viability) and IL-1alpha measurement. This allowed classification of corrosive chemicals (OECD guideline 431) and those likely to be severe irritants. If the chemical was found to be corrosive or a severe irritant, no further experimental work was performed, otherwise a second experiment was performed using three further exposure times (same endpoints). The second experiment provided information on whether the chemical was likely to be a moderate/mild irritant. If the chemical was negative following both experiments, it was predicted as non-corrosive/non-irritating. A total of 12 chemicals were tested in the irritation or combined assay (five non-irritants, seven irritants). Specificity (% non-irritants concurring with EU classification) was 60% (MTT) and 100% (MTT+IL-1alpha). Sensitivity (% irritants concurring with EU classification) was 86% (MTT) and 86% (MTT+IL-1alpha). Accuracy (% chemicals correctly identified) was 75% (MTT) and 92% (MTT+IL-1alpha).     

Dermal microdialysis of inflammatory markers induced by aliphatic hydrocarbons in rats

In the present study we made an attempt to understand the skin irritation cascade of selected aliphatic hydrocarbons using microdialysis technique. Microdialysis probes were inserted into dermis in the dorsal skin of hairless rats. After 2 h of probes insertion, occlusive dermal exposure (2 h) was carried out with 230 μl of nonane, dodecane and tetradecane, using Hill top chambers^®. Inflammatory biomarkers such as substance P (SP), α-melanocyte stimulating hormone (α-MSH) Interleukin 6 (IL-6) and prostaglandin E2 (PGE₂) were analyzed in the dialysis samples by enzyme immunoassay (EIA). SP, α-MSH and IL6 were released in significant amounts following the dermal exposure of nonane and dodecane, whereas tetradecane did not induce any of these markers in significant amounts compared to control. Nonane increased the PGE₂ levels in significant amounts within 2 h of chemical exposure compared to dodecane and tetradecane. IL-6 response was found to be slow and 2–3-fold increase in IL-6 levels was observed after 5 h following nonane and dodecane application. The magnitude of skin irritation exerted by all three chemicals was in the order of nonane ≥ dodecane ≥ tetradecane. The results demonstrate that microdialysis can be used to measure the inflammatory biomarkers in the skin irritation studies and irritation response of chemicals was quantifiable by this method. In conclusion, microdialysis was found to be an excellent tool to measure several inflammatory biomarkers as a function of time after dermal exposures with irritant chemicals.     

In vitro skin irritation testing on reconstituted human epidermis: reproducibility for 50 chemicals tested with two protocols.

Since it is of high importance to establish the skin irritation potential of industrial chemicals, toxicologists developed tests on various skin models. Most test data come from the rabbit Draize test, but its reproducibility is questionable. Some human in vivo test data exist, but they concern only few compounds. The emergence of new tools such as reconstituted human skin tissues offers a promising future to alternative methods. We describe here two in vitro skin irritation test protocols performed on reconstituted human epidermis. One is a direct topical application test and the other an in vitro patch test. Both protocols were performed using multiple endpoint analysis including cell viability (MTT reduction), histology, and IL-1alpha release. Fifty chemicals were tested: 20 compounds were used in the ECVAM pre-validation study and 30 products were previously tested in a human in vivo patch test. These in vitro skin irritation tests have not only the advantages of enhanced convenience and of reduced costs, but a good reproducibility is observed by endpoint, and by compound. A prediction model is proposed to classify the chemicals as irritant or non-irritant, and the results are compared to available rabbit and human data. We do not wish to overgeneralize from these 50 compounds; but, instead suggest that this data set be extensively extended to include chemicals of varying physico-chemical properties.     

Risk assessment for consumer exposure to toluene diisocyanate (TDI) derived from polyurethane flexible foam

Polyurethanes (PU) are polymers made from diisocyanates and polyols for a variety of consumer products. It has been suggested that PU foam may contain trace amounts of residual toluene diisocyanate (TDI) monomers and present a health risk. To address this concern, the exposure scenario and health risks posed by sleeping on a PU foam mattress were evaluated. Toxicity benchmarks for key non-cancer endpoints (i.e., irritation, sensitization, respiratory tract effects) were determined by dividing points of departure by uncertainty factors. The cancer benchmark was derived using the USEPA Benchmark Dose Software. Results of previous migration and emission data of TDI from PU foam were combined with conservative exposure factors to calculate upper-bound dermal and inhalation exposures to TDI as well as a lifetime average daily dose to TDI from dermal exposure. For each non-cancer endpoint, the toxicity benchmark was divided by the calculated exposure to determine the margin of safety (MOS), which ranged from 200 (respiratory tract) to 3 × 10⁶ (irritation). Although available data indicate TDI is not carcinogenic, a theoretical excess cancer risk (1 × 10⁻⁷) was calculated. We conclude from this assessment that sleeping on a PU foam mattress does not pose TDI-related health risks to consumers.     

Evaluation of eye and skin irritation of arginine-derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays

Arginine-derivative surfactants constitute a novel class of surfactants, which can be regarded as an alternative to conventional surfactants. Prior to human exposure, it is necessary to assess their irritation potential. The classical in vivo evaluation of the irritancy potential via the Draize test has been extensively criticized. In that regard, a great number of in vitro alternatives have been developed. Erythrocytes were chosen as the target cells for eye irritation assessment and hemolysis and hemoglobin denaturation were selected as appropriate endpoints. For skin irritancy assessment, the keratinocyte cell line NCTC 2544 was used and different in vitro endpoints were measured: two cytotoxicity assays (NRU and MTT) and the synthesis of the proinflammatory cytokine IL-1alpha. The eye and skin Draize tests were also performed for comparative purposes. The results point out that, according to in vivo and in vitro assays, the new arginine-derivative surfactants have lower eye and skin irritation potential than the synthetic surfactant SDS. Furthermore, in vitro methods were also able to detect differences in irritancy among the new surfactants not noticeable by the Draize tests, indicating that in vitro methods can be more sensitive than the in vivo test, offering the opportunity to detect subtle differences in irritancy.     

United States regulatory requirements for skin and eye irritation testing

Purpose: Eye and skin irritation test data are required or considered by chemical regulation authorities in the United States to develop product hazard labelling and/or to assess risks for exposure to skin- and eye-irritating chemicals. The combination of animal welfare concerns and interest in implementing methods with greater human relevance has led to the development of non-animal skin- and eye-irritation test methods. To identify opportunities for regulatory uses of non-animal replacements for skin and eye irritation tests, the needs and uses for these types of test data at U.S. regulatory and research agencies must first be clarified.

Methods: We surveyed regulatory and non-regulatory testing needs of U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) agencies for skin and eye irritation testing data. Information reviewed includes the type of skin and eye irritation data required by each agency and the associated decision context: hazard classification, potency classification, or risk assessment; the preferred tests; and whether alternative or non-animal tests are acceptable. Information on the specific information needed from non-animal test methods also was collected.

Results: A common theme across U.S. agencies is the willingness to consider non-animal or alternative test methods. Sponsors are encouraged to consult with the relevant agency in designing their testing program to discuss the use and acceptance of alternative methods for local skin and eye irritation testing.

Conclusions: To advance the implementation of alternative testing methods, a dialog on the confidence of these methods to protect public health and the environment must be undertaken at all levels.     

Comparison of in vitro and in vivo human skin responses to consumer products and ingredients with a range of irritancy potential.

Human skin equivalent cultures were investigated as possible pre-clinical skin irritation screens to aid safety assessments for chemicals and product formulations, and to facilitate design of safe and efficient human studies. In vitro responses in human skin equivalent cultures were compared directly to in vivo human skin responses from historic or concurrent skin tests for representative chemicals and products, including surfactants, cosmetics, antiperspirants, and deodorants. The in vivo data consisted of visual scores (i.e., erythema and edema) from skin-patch tests and diary accounts of skin irritation from product-use studies. In the in vitro studies, cornified, air-interfaced human skin cultures (EpiDerm) were evaluated using methods designed to parallel human clinical protocols with topical dosing of neat or diluted test substances to the stratum corneum surface of the skin cultures. The in vitro endpoints have previously been shown to be relevant to human skin irritation in vivo, including the MTT metabolism assay of cell viability, enzyme release (lactate dehydrogenase and aspartate aminotransferase), and inflammatory cytokine expression (Interleukin-1alpha). For surfactants, dose-response curves of MTT cell-viability data clearly distinguished strongly-irritating from milder surfactants and rank-ordered irritancy potential in a manner similar to repeat-application (3x), patch-test results. For the antiperspirant and deodorant products, all the in vitro endpoints correlated well with consumer-reported irritation (r, 0.75-0.94), with Interleukin-1alpha (IL-1alpha) release, showing the greatest capacity to distinguish irritancy over a broad range. IL-1alpha release also showed the best prediction of human skin scores from 14-day cumulative irritancy tests of cosmetic products. These results confirm the potential value of cornified human skin cultures as in vitro pre-clinical screens for prediction of human skin irritation responses. A preliminary report of these results has been published.     

Intralaboratory validation of alternative endpoints in the murine local lymph node assay for the identification of contact allergic potential: primary ear skin irritation and ear-draining lymph node hyperplasia induced by topical chemicals

We validated a two-tiered murine local lymph node assay (LLNA) with a panel of standard contact (photo)allergens and (photo)irritants with the aim of improving the discrimination between contact (photo) allergenic potential and true skin (photo)irritation potential. We determined ear weights to correlate chemical-induced skin irritation with the ear-draining lymph node (LN) activation potential. During tier I LLNAs, a wide range of concentrations were applied on three consecutive days to the dorsum of both ears. Mice were exposed to UVA light immediately after topical application to determine the photoreactive potential of some test chemicals. Mice were killed 24 h after the last application to determine ear and LN weights and LN cell counts. It was possible to classify the tested chemicals into three groups according to their threshold concentrations for LN activation and skin irritation: (1) chemicals with a low LN activation potential and no or very low skin irritation potential; (2) chemicals with a marked LN activation potential higher than a distinct skin irritation potential; and (3) chemicals with LN activation potential equal to or lower than their skin irritation potential. Group 1 consisted only of contact allergens, indicating that LN activation in the absence of skin irritation points to a contact allergenic activity. Since groups 2 and 3 comprised irritants and contact allergens, a tier II LLNA protocol was used to finally differentiate between true irritants and contact allergens. Briefly, mice were pretreated with mildly to moderately irritating concentrations of the chemical to the shaved back and after 12 days were challenged on the ears as described above in order to elicit a contact allergenic response in the ear skin and the ear-draining LN. With this approach, tier II LLNAs have to be conducted only in cases for which skin irritation potential is in the range of LN activation potential and no structure-activity relationship data indicating a contact allergenic hazard are available.     

The effect of occlusive and unocclusive exposure to xylene and benzene on skin irritation and molecular responses in hairless rats

Aromatic hydrocarbons readily penetrate the skin on dermal exposure, leading to irritation, inflammation and cytotoxicity. The effects of short-term occlusive and long-term unocclusive dermal exposure to benzene and xylene on the skin irritation response (transepidermal water loss (TEWL), skin moisture content and erythema) and cytokine/chemokine expression (interleukin-1 (IL-1), tumor necrosis factor- (TNF-) and monocyte chemoattractant protein-1 (MCP-1)) were investigated in hairless rats. Occlusive dermal exposure was carried out with 230 L of the chemicals for 1 h using Hill top chambers. In unocclusive dermal exposure, 15 L of the chemicals were applied to the skin every 2 h, for 8 h a day, for 4 days. The occlusive dermal exposure revealed a clear difference in the TEWL and erythema response of these chemicals (xylene>benzene) whereas unocclusive exposure revealed similar TEWL and erythema scores for both benzene and xylene. The expression of IL-1 was elevated 2.5- and 3.8-fold in response to occlusive and unocclusive exposure, respectively, vs control (P<0.01) for both the chemicals (benzene and xylene). Similarly, TNF- levels were elevated about 2.4- and 6.0-fold as a result of occlusive and unocclusive exposure, respectively, vs control (P<0.01). These results show that unocclusive exposure induced significantly higher TNF- expression than occlusive exposure (P<0.05). The MCP-1 expression in blood was slightly elevated compared with the control group, but this increase was not statistically significant (P>0.05). Similarly, MCP levels in skin were increased approximately 1.7- and 1.8-fold by occlusive and unocclusive exposure, respectively, compared with the control group (P<0.05). Our study demonstrates that the skin irritation profiles of benzene and xylene are similar and unocclusive long-term exposure to small amounts of these chemicals can induce more skin irritation and cytokine response than occlusive exposure.     

Quantitative risk assessment for skin sensitization: Success or failure?

Skin sensitization is unique in the world of toxicology. There is a combination of reliable, validated predictive test methods for identification of skin sensitizing chemicals, a clearly documented and transparent approach to risk assessment, and effective feedback from dermatology clinics around the world delivering evidence of the success or failure of the hazard identification/risk assessment/management process. Recent epidemics of contact allergy, particularly to preservatives, have raised questions of whether the safety/risk assessment process is working in an optimal manner (or indeed is working at all!). This review has as its focus skin sensitization quantitative risk assessment (QRA). The core toxicological principles of QRA are reviewed, and evidence of use and misuse examined. What becomes clear is that skin sensitization QRA will only function adequately if two essential criteria are met. The first is that QRA is applied rigourously, and the second is that potential exposure to the sensitizing substance is assessed adequately. This conclusion will come as no surprise to any toxicologist who appreciates the basic premise that “risk = hazard x exposure”. Accordingly, use of skin sensitization QRA is encouraged, not least because the essential feedback from dermatology clinics can be used as a tool to refine QRA in situations where this risk assessment tool has not been properly used.     

吲哚美辛乳剂对皮肤刺激性和过敏性研究

目的考察吲哚美辛乳剂的皮肤刺激性和皮肤过敏性,为临床安全用药提供依据。方法采用新西兰白兔进行皮肤刺激性实验,分为完整皮肤组和破损皮肤组,每组8只。采用同体左、右侧自身对比法,左侧给予吲哚美辛乳剂0.5g,右侧给予等量空白乳剂,连续给药14d。实验期间观察皮肤刺激反应,每组6只于末次给药后72h处死,取皮肤做组织病理检查,剩余2只于末次给药后14d取皮肤做病理检查。采用豚鼠进行皮肤过敏实验,于第0、7、14天给予吲哚美辛0.5g局部诱导,第28天在未给药豚鼠的肋腹部皮肤给予吲哚美辛0.4g以局部激发。结果吲哚美辛乳剂对兔正常皮肤和破损皮肤均有轻微刺激性,但停药后可恢复,对豚鼠皮肤无过敏反应。结论吲哚美辛乳剂在临床上不宜长期使用,需重点观察皮肤刺激性。     

An in vitro human skin test for assessing sensitization potential

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold‐standard method for identification and characterization of skin‐sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in‐vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro‐haptens, respiratory sensitizers, non‐sensitizing chemicals (including skin‐irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non‐sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in‐vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process. Copyright © 2015 John Wiley & Sons, Ltd.     

Rat epidermal keratinocyte organotypic culture (ROC) as a model for chemically induced skin irritation testing

The potential of rat epidermal keratinocyte (REK) organotypic culture (ROC) with proper stratum corneum barrier as a model for screening skin irritants was evaluated. The test chemicals were selected from ECETOC database (1995) and the observed in vitro irritation potential was compared to ECETOC in vivo primary irritation index (PII), to EU risk phrases, and to the harmonized OECD criteria. Chemicals were applied onto the stratum corneum surface of ROC for 30 min and samples were taken from the underlying medium at 4 and 8 h after exposure. Cell membrane integrity (determined by LDH assay) and pro-inflammatory effect (determined by IL-1alpha release) were verified at both time points and correlated to PII values. The best correlation (R(2) = 0.831) was seen with LDH leakage test. Based on obtained data, chemicals were classified according to criteria defined by EU and OECD. From 12 chemicals, only two were incorrectly classified according to OECD criteria when using LDH leakage and IL-1alpha release as irritation markers. At the end of experiment, chemical-treated ROC cultures were fixed and histological changes were assessed. Typical signs for irritation were lightly stained cytoplasm, condensed nuclei, cellular vacuolization, eosinophilic cytoplasms, and blebbing. These irritation effects of chemicals were graded visually into four classes (A-D). The extent of morphological perturbations of the cultures mostly correlated with PII. The present results indicate the validity of the ROC model in predicting skin irritation potential of chemicals and show that the use of set of irritation markers with different mechanistic responses gives more information on irritation than if only one marker was used.     

Effect of JP-8 jet fuel on molecular and histological parameters related to acute skin irritation

Organic chemicals such as jet fuels and solvents can cause skin irritation after dermal exposure. The molecular responses to these chemicals resulting in acute irritation are not understood well enough to establish safe exposure limits. Male F-344 rats were dermally exposed to JP-8 jet fuel for 1 h using Hill Top Chambers. Whole skin samples were collected at 0, 1, 2, 4, and 6 h after the beginning of the exposures, homogenized, and analyzed for interleukin (IL)-1alpha and inducible nitric oxide synthase (iNOS) protein and nitrite levels. IL-1alpha levels (determined by ELISA) ranged from approximately 11 to 34% above the 0-h samples over the observed time period. At 1 and 2 h, significantly higher (p < 0.05) levels of IL-1alpha were detected when compared to the 0-h samples. Western blot analysis revealed significantly higher (p < 0.05) levels of iNOS at 4 and 6 h compared to 0-h samples. Increases in IL-1alpha and iNOS expression were also observed in the skin immunohistochemically. Nitrite concentrations in skin samples were measured to estimate nitric oxide production. Although nitrite concentrations in the skin increased approximately 6-27% above the 0-h samples over the observed time period, no significant changes in nitrite levels were detected. Pathological changes in the skin following JP-8 exposure were evaluated histologically. Increased numbers of granulocytes were observed infiltrating the skin at 2 h and were more prominent by 6 h. These data show that a 1-h exposure to JP-8 results in a local inflammatory response, which can be detected by changes in molecular and histological parameters.     

Dose response assessment for effects of acute exposure to methyl isothiocyanate (MITC)

The preplant fumigants, metam-sodium, metam-potassium, and dazomet undergo decomposition to the biocide methyl isothiocyanate (MITC) in moist soils. Since MITC vapor can migrate from its site of application, we developed an estimate of health protective concentrations for airborne exposures to MITC that prevents effects among bystanders near treated agricultural fields. Our findings show that, at concentrations of environmental relevance, MITC most likely acts via stimulation of the trigeminal nerve, which mediates sensory irritation in the eyes and nose. Several lines of evidence support the conclusion that sensory irritation of the eyes is the most sensitive effect relevant for health risk assessment arising from short-term MITC exposures. The outcome of a clinical study that included sensitive individuals and measured multiple ocular responses to irritation (e.g., perceived irritation, tearing, and blinking of the eyes) is consistent with this proposed mode of action, as are experimental animal data. Databases and studies by the California Department of Pesticide Regulation (CDPR) show that, in accidental exposures, human eye irritation is consistently the most sensitive endpoint at low-modeled acute exposure and is often the most sensitive endpoint from acute exposures of unknown, but likely higher, concentrations. Based upon benchmark concentration lower limits from the clinical study and consideration of uncertainties, health protective concentrations of MITC were estimated as 0.2 ppm for 4 h of exposure and 0.8 ppm for 14-min of exposure.     

The OSIRIS Weight of Evidence approach: ITS for skin sensitisation

Within the EU FP6 project OSIRIS approaches to Integrated Testing Strategies (ITSs) were developed, with the aim to facilitate the use of non-test and non-animal testing information in regulatory risk assessment of chemicals. This paper describes an analytical Weight-of-Evidence (WoE) approach to an ITS for the endpoint of skin sensitisation. It specifically addresses the European chemicals legislation REACH, but the concept is readily applicable to ITS and WoE procedures in other regulatory frameworks, and for other toxicological endpoints. Bayesian statistics are applied to estimate the reliability of a conclusion on the sensitisation potential of a chemical, combining evidence from different information sources such as QSAR model predictions, in vitro and in vivo test results. The methodology allows for adaptation of the weight of individual information sources to account for the different levels of reliability of the individual ITS components. The calculated reliability of the WoE conclusion gives an objective, transparent and reproducible measure to decide if the information requirements for data evaluation are satisfied. Furthermore, in case the WoE is not sufficient, it gives the possibility to evaluate a priori if and how it will be possible to fulfil the information requirements with additional tests and/or model predictions.