Hitoshi Sakaguchi

Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line.

It has been known that dendritic cells (DCs) including Langerhans cells (LCs) play a critical role in the skin sensitization process. Many attempts have been made to develop in vitro sensitization tests that employ DCs derived from peripheral blood mononuclear cells (PBMC-DC) or CD34+ hematopoietic progenitor cells (CD34+ HPC) purified from cord blood or bone marrow. However, the use of the DCs in in vitro methods has been difficult due to the nature of these cells such as low levels in the source and/or donor-to-donor variability. In our studies, we employed the human monocytic leukemia cell line, THP-1, in order to avoid some of these difficulties. At the start, we examined whether treatment of the cells with various cytokines could produce DCs from THP-1. Treatment of THP-1 cells with cytokines such as GM-CSF, IL-4, TNF-alpha, and/or PMA did induce some phenotypic changes in THP-1 cells that were characteristic of DCs. Subsequently, responses to a known sensitizer, dinitrochlorobenzene (DNCB), and a non-sensitizer, dimethyl sulfoxide (DMSO) or sodium lauryl sulfate (SLS), on the expression of co-stimulatory molecules, CD54 and CD86, were examined between the naive cells and the cytokine-treated cells. Interestingly, the naive THP-1 cells responded only to DNCB and the response to the sensitizer was more distinct than cytokine-treated THP-1 cells. Similar phenomena were also observed in the human myeloid leukemia cell line, KG-1. Furthermore, with treatment of DNCB, naive THP-1 cells showed augmented expression of HLA, CD80 and secretion of IL-1 beta. The response of THP-1 cells to a sensitizer was similar to that of LCs/DCs. Upon demonstrating the differentiation of monocyte cells in our system, we then evaluated a series of chemicals, including known sensitizers and non-sensitizers, for their potential to augment CD54 and CD86 expression on naive THP-1 cells. Indeed, known sensitizers such as PPD and 2-MBT significantly augmented CD54 and CD86 expression in a dose-dependent manner while non-sensitizers, such as SLS and methyl salicylate (MS), did not. To note, the metal allergens such as (NH(4))(2)[PtCl(4)], NiSO(4) and CoSO(4) augmented significantly only CD54 expression. Taking advantage of a cultured cell line, measurement of the co-stimulatory molecules, CD54 and CD86, on naive THP-1 cells following chemical exposure shows promise for the development of a simple, short-term in vitro sensitization test.

Categorization of chemicals according to their relative human skin sensitizing potency.

Although adoption of skin sensitization in vivo assays for hazard identification is likely to be successful in the next few years, this does not replace their use in potency prediction. Notably, measurement of potency of skin sensitizers in the local lymph node assay has been important. However, this local lymph node assay potency measure has not been formally assessed against a range of substances of known human sensitizing potential, because the latter is lacking. Accordingly, criteria for human data have been established that characterize 6 categories of human sensitizing potency, with 1 the most potent and 5 the least potent; category 6 represents true nonsensitizers. The literature has been searched, and 131 chemicals assigned into these categories according to their intrinsic potency judged only by the available human information. The criteria and data set generated provide a basis for examination of the capacity of nonanimal approaches for the determination of human sensitization potency.

Data integration of non-animal tests for the development of a test battery to predict the skin sensitizing potential and potency of chemicals.

Recent changes in regulatory restrictions and social views against animal testing have accelerated development of reliable alternative tests for predicting skin sensitizing potential and potency of many chemicals. Lately, a test battery integrated with different in vitro tests has been suggested as a better approach than just one in vitro test for replacing animal tests. In this study, we created a dataset of 101 test chemicals with LLNA, human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and in silico prediction system. The results of these tests were converted into scores of 0-2 and the sum of individual scores provided the accuracy of 85% and 71% for the potential and potency prediction, compared with LLNA. Likewise, the straightforward tiered system of h-CLAT and DPRA provided the accuracy of 86% and 73%. Additionally, the tiered system showed a higher sensitivity (96%) compared with h-CLAT alone, indicating that sensitizers would be detected with higher reliability in the tiered system. Our data not only demonstrates that h-CLAT can be part of a test battery with other methods but also supports the practical utility of a tiered system when h-CLAT and DPRA are the first screening methods for skin sensitization.

Identifying and characterizing chemical skin sensitizers without animal testing: Colipa's research and method development program

The sensitizing potential of chemicals is usually identified and characterized using one of the available animal test methods, such as the mouse local lymph node assay. Due to the increasing public and political concerns regarding the use of animals for the screening of new chemicals, the Colipa Skin Tolerance Task Force collaborates with and/or funds research groups to increase and apply our understanding of the events occurring during the acquisition of skin sensitization. Knowledge gained from this research is used to support the development and evaluation of novel alternative approaches for the identification and characterization of skin sensitizing chemicals. At present one in chemico (direct peptide reactivity assay (DPRA)) and two in vitro test methods (cell based assays (MUSST and h-CLAT)) have been evaluated within Colipa inter-laboratory ring trials and accepted by the European Centre for the Validation of Alternative Methods (ECVAM) for pre-validation. Data from all three test methods will be used to support the development of testing strategy approaches for skin sensitizer potency prediction. The replacement of the need for animal testing for skin sensitization risk assessment is viewed as ultimately achievable and the next couple of years should set the timeline for this milestone.

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test – human cell line activation test (h-CLAT)

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20–95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65–90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of “CD86 ≥ 150 or CD54 ≥ 200” resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells.

Using SIRC (rabbit corneal cell line) cells, we developed an alternative eye irritation test: the short time exposure (STE) test. This STE test is a cytotoxicity test using physiological saline or mineral oil as the test solvent. Evaluation exposure time is short (5 min), which is similar to actual exposure situations, and uses the cell viability (CV) at a constant concentration as the endpoint for irritation potential. First, in order to confirm the usefulness of this STE test in assessing eye irritation potential of chemicals, 51 raw materials were tested and the correlation between CV in the STE test and the eye irritation score in the Draize test was examined. For the undiluted raw materials tested in the Draize test, the 5% test concentration in the STE test gave irritation classes that correlated well with the irritation classes from the Draize test (accuracy: 89.6%). For those materials tested as a 10% solution in the Draize test, STE irritation classes with 0.05% test concentration corresponded well with the Draize irritation classes (accuracy: 80.0%). Next, using the cell viabilities at these two concentrations, the STE prediction model (PM) was developed. A score of 1 or 2 was given for the results from each tested concentration in the STE test and Draize test. The scores from each test were then summed to yield a 3-level (Rank 1: minimally irritant, Rank 2: moderate irritant, Rank 3: severe irritant) eye irritation potential classification. Rank classification in the STE test showed a good correlation mostly to that in the Draize test (irritation class correspondence rate: 70.2%, but after exclusion of data of alcoholic materials, the rate was 91.7%). In most cytotoxicity test, the cytotoxicity of acids and amines is generally underestimated due the use of medium as the solvent. This is the result of the buffering capacity of the media. On the other hand, the STE test could predict the eye irritation potential by evaluating the chemical with a 5% test concentration. Eleven water insoluble materials such as toluene, octanol, and hexanol could be evaluated by using mineral oil as test solvent in the STE test. The STE test demonstrated itself to be simple, promising, have great potential, be of value, and to be an easily standardized alternative eye irritation test.

Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency.

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI=3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI=150 for CD86 (EC150), and for CD54 (EC200) with LLNAs EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R=0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 μg/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay.

Predictive performance for human skin sensitizing potential of the human cell line activation test (h-CLAT)

Background. Recent changes in regulatory restrictions and social opposition to animal toxicology experiments have driven the need for reliable in vitro tests for predicting the skin sensitizing potentials of a wide variety of industrial chemicals. Previously, we developed the human cell line activation test (h‐CLAT) as a cell‐based assay to predict the skin sensitizing potential of chemicals, and showed the correspondence between the h‐CLAT and the murine local lymph node assay results.

Validation study of the Short Time Exposure (STE) test to assess the eye irritation potential of chemicals.

Short time exposure (STE) test is a cytotoxicity test in SIRC cells (rabbit corneal cell line) that assesses eye irritation potential following a 5-min chemical exposure. This validation study assessed transferability, intra- and inter-laboratory reproducibility, and predictive capacity of STE test in five laboratories (supported by Japanese Society for Alternatives to Animal Experiments). Sodium lauryl sulfate, calcium thioglycolate, and Tween 80 were evaluated, in triplicate, using 5%, 0.5%, and 0.05% concentrations in physiological saline, to confirm transferability. Good transferability was noted when similar mean relative viabilities and rank classifications were obtained in all five laboratories and were comparable to data from test method developing laboratory. Good intra- and inter-laboratory reproducibility was obtained with four assay controls (three solvents and one positive control), and four assay controls and 25 chemicals, respectively. STE irritation category based on relative viability of a 5% solution of 25 blinded test chemicals showed good correlation with Globally Harmonized System (GHS) categories (NI; I: Cat. 1 and 2). The STE prediction model, using relative viability of the 5% and 0.05% solutions, provided an irritation rank (1, 2, or 3) that had a good correlation (above 80%), or predictive capacity, with GHS irritation ranks in all laboratories. Based on these findings, the STE test is a promising alternative eye irritation test that could be easily standardized.