We evaluated the inhibitory effects of nano-sized liposomal particles, hybrid liposomes (HLs), on the growth of breast cancer stem cells (BCSCs), and the effectiveness of soft agar colony formation assay for discriminating BCSCs. The flow cytometric (FCM) analysis of mouse-derived breast cancer (4T1-Luc) cells, using CD44v marker, inferred they contain 70% – 90% of BCSCs. In 4T1-Luc cells, the proportion of BCSCs remarkably decreased after adding HLs and was almost constant after adding doxorubicin (DOX). The 50% inhibitory concentration (IC₅₀) of DOX was 0.121 μM for 4T1-Luc cells. The 4T1-Luc cell number decreased after adding 5 μM DOX, without reducing BCSCs. After adding HL at concentrations above the IC₅₀ (99.6 μM) for 4T1-Luc cells, BCSCs gradually decreased to 37% of their concentration at 300 μM.

Furthermore, a reduction in BCSC proportion in HL-treated 4T1-Luc cells was observed using the soft agar colony formation method. Based on the HL-treated concentration, the live cell number significantly decreased and cell-survival was maintained with DOX treatment. Fusion and accumulation of the fluorescent phospholipid (NBDPC)-labeled HLs in these cells were analyzed using FCM and confocal laser scanning microscopy.

These results indicate that HL is more effective in suppressing the growth of BCSCs than DOX. The soft agar colony formation assay, which measures the tumorigenicity of cells in vitro, may be a promising discrimination method for the BCSCs, further reducing animal testing.

Respiratory sensitization induced by chemicals is a critical occupational and public health issue because it is associated with allergic asthma or other pulmonary symptoms. However, there are no validated test methods to identify the potential to cause respiratory sensitization. In this study, to investigate the behavior of respiratory sensitizers in a skin sensitization test method, 14　respiratory sensitizers were tested with the human cell line activation test (h-CLAT). h-CLAT was positive for seven of the 14 respiratory sensitizers, including all four diisocyanate used industrially as polyurethane materials. Furthermore, for three acid anhydrides negative in h-CLAT, we conducted a modified h-CLAT (a short-time exposure method using liquid paraffin), and phthalic anhydride proved positive. Among the seven h-CLAT negatives, at least four chemicals were positive in the direct peptide reactivity assay (DPRA - in chemico skin sensitization test method listed in OECD TG 442C). Therefore, to identify the potential to cause respiratory sensitization, it is necessary to not only the application to in vitro skin sensitization test based on the common key events but also the combination of these existing skin sensitization tests with the new approach methods, which is developed based on the specific mechanism of chemical respiratory allergy.

Receptor binding tests for various receptors of substances are widely employed to identify drug candidates and predict the biological effects of chemical substances. Here, the results of binding to androgen receptor (AR) reported in the validation study of OECD TG458 and Hansen solubility parameter (HSP) values of the test substance were compared and examined by the Hansen sphere method, thus giving potential HSPs considered to correspond to the AR binding domain of agonists and antagonists. Our results suggest that the agonist/antagonist AR binding property could be predicted with high accuracy using our calculated potential HSPs. To our knowledge, this is the first study to report the application of HSPs to determine the safety of substances and protein structure–activity relationship.