Kazutoshi Saito

Development of a new in vitro skin sensitization assay (Epidermal Sensitization Assay; EpiSensA) using reconstructed human epidermis.

Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.

Binary test battery with KeratinoSens™ and h-CLAT as part of a bottom-up approach for skin sensitization hazard prediction

ABSTRACT Skin sensitization is one of the key safety endpoints for chemicals applied directly to the skin. Several integrated testing strategies (ITS) using multiple non‐animal test methods have been developed to accurately evaluate the sensitizing potential of chemicals, but there is no regulatory‐accepted ITS to classify a chemical as a non‐sensitizer. In this study, the predictive performance of a binary test battery with KeratinoSens™ and h‐CLAT compared to the local lymph node assay (LLNA) and human data was examined using comprehensive dataset of 203 chemicals. When two negative results indicate a non‐sensitizer, the binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data. Taking into account the predictive limitations (i.e. high log Kow, pre‐/pro‐haptens and acyl transfer agents (or amine‐reactive)), the binary test battery had extremely high sensitivity comparable to that of the 3 out of 3 ITS where three negative results of the DPRA, KeratinoSens™ and h‐CLAT indicate a non‐sensitizer. Therefore, the data from KeratinoSens™ or h‐CLAT may provide partly redundant information on the molecular initiating event derived from DPRA. Taken together, the binary test battery of KeratinoSens™ and h‐CLAT could be used as part of a bottom‐up approach for skin sensitization hazard prediction. HIGHLIGHTSPredictivity of a binary test battery with KeratinoSens™ and h‐CLAT compared to the LLNA and human data was examined.The binary test battery provided sensitivity of 93.4% or 94.4% compared with the LLNA or human data, respectively.KeratinoSens™ or h‐CLAT may provide partly redundant information on the molecular initiating event derived from DPRA.The binary test battery could be used as part of a bottom‐up approach for skin sensitization hazard prediction.

Effects of dietary alpha-linolenic acid-enriched diacylglycerol oil on embryo/fetal development in rats

Recent studies suggest that diets supplemented with alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil provide potential health benefits in preventing or managing obesity. However, available safety information about reproductive and developmental toxicities of ALA-DAG oil is limited. This study was conducted to clarify the effect, if any, of ALA-DAG oil on embryo-fetal development, following maternal exposure during the critical period of major organogenesis. ALA-DAG oil was administered via gavage to pre-mated female Sprague Dawley rats from gestation day 6 through 19, at dose levels of 0, 1.25, 2.5, and 5.0 mL/kg/day (equivalent to 0, 1149, 2325, and 4715 mg/kg/day, respectively), with total volume adjusted to 5 mL/kg/day with rapeseed oil. All females survived to the scheduled necropsy. There were no treatment-related changes in clinical or internal findings, maternal body weights, feed consumption, intrauterine growth, survival, and number of implantations. No ALA-DAG oil-related fetal malformations or developmental variations were noted. A maternal maximum tolerated dose for ALA-DAG oil could not be achieved in this study. Based on these results, a dose level of 5.0 mL/kg (4715 mg/kg/day), the highest dose tested, was considered as the no-observed-adverse-effect level (NOAEL) for both maternal and developmental toxicity.

A 90-day repeated-dose toxicity study of dietary alpha linolenic acid-enriched diacylglycerol oil in rats

ABSTRACT Diets supplemented with alpha‐linolenic acid (ALA)‐enriched diacylglycerol (DAG) oil—which mainly consists of oleic and linolenic, linoleic acids—have potential health benefits in terms of preventing or managing obesity. Although safety of DAG oil has been extensively investigated, toxicity of ALA‐DAG oil has not been well understood. Hence, the present study was conducted to clarify the potential adverse effects, if any, of ALA‐DAG oil in rats (10/sex/group) fed diets containing 1.375%, 2.75%, or 5.5% ALA‐DAG oil for 90 days. Compared to control rats fed rapeseed oil or ALA‐triacylglycerol oil (flaxseed oil), rats receiving ALA‐DAG oil did not reveal any toxicologically significant treatment‐related changes as evaluated by clinical signs, functional observational battery, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weight, necropsy and histopathology. The no observed adverse effect levels for dietary exposure to ALA‐DAG oil for male and female rats were 2916 and 3326mg/kg body weight/day, respectively, the highest dose tested. The findings from this study suggest that consumption of ALA‐DAG oil is unlikely to cause adverse effects. HIGHLIGHTSThis study was investigated to clarify the toxicological potential of ALA‐DAG oil for 90 days.ALA‐DAG oil did not reveal any toxicologically significant treatment‐related changes.The no observed adverse effect levels for male and female rats were 2916 and 3326mg/kg body weight/day, respectively.

Predictive performance of the Short Time Exposure test for identifying eye irritation potential of chemical mixtures.

The Short Time Exposure (STE) test is an in vitro eye irritation test based on the cytotoxicity in SIRC cells (rabbit corneal cell line) following a 5 min treatment of chemicals. This study evaluated the predictive performance of the STE test to identify the globally harmonized system (GHS) Not Classified category and other irritant categories (i.e., GHS Category 1 or 2) when used to test 40 chemical mixtures that included irritants. The STE test correctly identified 30 tested mixtures classified as GHS irritant categories and 5 out of 10 tested mixtures classified as GHS Not Classified. The sensitivity, specificity, positive predictivity, negative predictivity, and overall accuracy of the STE test were 100% (30/30), 50% (5/10), 86% (25/30), 100% (5/5), and 88% (35/40), respectively. These predictive performances were comparative to or greater than those in other in vitro eye irritation tests that have been accepted as test guideline by the Organisation for Economic Co-operation and Development. This suggests that the STE test has sufficient predictivity for identifying the eye irritation potential of chemical mixtures. Since no false negatives in this study were found, this indicates that the STE test is applicable as a part of the bottom-up approach.

Transferability and within- and between-laboratory reproducibilities of EpiSensA for predicting skin sensitization potential in vitro: A ring study in three laboratories: RING STUDY OF EPISENSA FOR PREDICTING SKIN SENSITIZATION POTENTIAL

The epidermal sensitization assay (EpiSensA) is an in vitro skin sensitization test method based on gene expression of four markers related to the induction of skin sensitization; the assay uses commercially available reconstructed human epidermis. EpiSensA has exhibited an accuracy of 90% for 72 chemicals, including lipophilic chemicals and pre−/pro‐haptens, when compared with the results of the murine local lymph node assay. In this work, a ring study was performed by one lead and two naive laboratories to evaluate the transferability, as well as within‐ and between‐laboratory reproducibilities, of EpiSensA. Three non‐coded chemicals (two lipophilic sensitizers and one non‐sensitizer) were tested for the assessment of transferability and 10 coded chemicals (seven sensitizers and three non‐sensitizers, including four lipophilic chemicals) were tested for the assessment of reproducibility. In the transferability phase, the non‐coded chemicals (two sensitizers and one non‐sensitizer) were correctly classified at the two naive laboratories, indicating that the EpiSensA protocol was transferred successfully. For the within‐laboratory reproducibility, the data generated with three coded chemicals tested in three independent experiments in each laboratory gave consistent predictions within laboratories. For the between‐laboratory reproducibility, 9 of the 10 coded chemicals tested once in each laboratory provided consistent predictions among the three laboratories. These results suggested that EpiSensA has good transferability, as well as within‐ and between‐laboratory reproducibility.