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Despite widespread and prolonged use of adult novelties, their health safety is not regularly tested or legally regulated. In the EU, adult novelties are subjected to the General Product Safety Directive, placing the burden of proof regarding safe products onto the manufacturers. The aim of our pilot study was to expand knowledge on potential application of in vitro methods for hazard prediction of extracts from final products. We subjected extracts of 20 adult novelties, purchased on the Czech market to toxicological tests including NRU cytotoxicity assay, sensitization tests DPRA and LuSens and the YES/YAS endocrine assay. Four samples produced cytotoxicity. Sensitization potential was recorded by DPRA (three samples) while the LuSens reported ten samples. Regarding endocrine disruption, three samples produced antiestrogen and antiandrogen effects. Six samples exhibited androgenic potential and one sample showed estrogenic potential. Positive results with possible health effects were recorded repeatedly for samples made of ABS, PVC and latex. The study has confirmed promising usefulness of our test methods combination with regard to safety testing of this type of consumer products. The results should be evaluated with care, however, the data bring added-value to the limited knowledge of mixture toxicology and are indicative for further testing.

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Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells / tissues of human origin.

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BACKGROUND@#Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bonedefects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelialgrowth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This studyassessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derivedmesenchymal stem/stromal cells (AT-MSCs). @*METHODS@#Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response.Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations forlong-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiationof AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogeneticprotein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secretedphosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4(KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. @*RESULTS@#PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose- and time dependent antiproliferativeeffects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic inductionon the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressedosteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. @*CONCLUSION@#PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelatedgenes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on theosteogenic differentiation of AT-MSCs.

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BACKGROUND@#Insufficient vascularization hampers bone tissue engineering strategies for reconstructing large bonedefects. Delivery of prolyl-hydroxylase inhibitors (PHIs) is an interesting approach to upregulate vascular endothelialgrowth factor (VEGF) by mimicking hypoxic stabilization of hypoxia-inducible factor-1alpha (HIF-1a). This studyassessed two PHIs: dimethyloxalylglycine (DMOG) and baicalein for their effects on human adipose tissue-derivedmesenchymal stem/stromal cells (AT-MSCs). @*METHODS@#Isolated AT-MSCs were characterized and treated with PHIs to assess the cellular proliferation response.Immunostaining and western-blots served to verify the HIF-1a stabilization response. The optimized concentrations forlong-term treatment were tested for their effects on the cell cycle, apoptosis, cytokine secretion, and osteogenic differentiationof AT-MSCs. Gene expression levels were evaluated for alkaline phosphatase (ALPL), bone morphogeneticprotein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor A (VEGFA), secretedphosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). In addition, stemness-related genes Kruppel-like factor 4(KLF4), Nanog homeobox (NANOG), and octamer-binding transcription factor 4 (OCT4) were assessed. @*RESULTS@#PHIs stabilized HIF-1a in a dose-dependent manner and showed evident dose- and time dependent antiproliferativeeffects. With doses maintaining proliferation, DMOG and baicalein diminished the effect of osteogenic inductionon the expression of RUNX2, ALPL, and COL1A1, and suppressed the formation of mineralized matrix. Suppressedosteogenic response of AT-MSCs was accompanied by an upregulation of stemness-related genes. @*CONCLUSION@#PHIs significantly reduced the osteogenic differentiation of AT-MSCs and rather upregulated stemnessrelatedgenes. PHIs proangiogenic potential should be weighed against their longterm direct inhibitory effects on theosteogenic differentiation of AT-MSCs.

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Vasculogenesis and angiogenesis are the processes by which new blood vessels are formed. We have developed a serum-free human adipose stromal cell and umbilical cord vein endothelial cell based vasculogenesis/angiogenesis test. In this study, the test was validated in our GLP laboratory following the OECD Guidance Document 34 [1] using erlotinib, acetylic salicylic acid, levamisole, 2-methoxyestradiol, anti-VEGF, methimazole, and D-mannitol to show its reproducibility, repeatability, and predictivity for humans. The results were obtained from immunostained tubule structures and cytotoxicity assessment. The performance of the test was evaluated using 26 suspected teratogens and non-teratogens. The positive predictive value was 71.4% and the negative predictive value was 50.0%, indicating that inhibition of vasculogenesis is a significant mechanism behind teratogenesis. In conclusion, this test has great potential to be a screening test for prioritization purposes of chemicals and to be a test in a battery to predict developmental hazards in a regulatory context.

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The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

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The effects of tamoxifen, toremifene and chloroquine on the phagocytosis of rod outer segments by retinal pigment epithelium were evaluated in human retinal pigment epithelial cell line D407 and pig retinal pigment epithelial cell culture. Retinal pigment epithelial cells were exposed to different concentrations of tamoxifen (1-20 microM), toremifene (1-20 microM) and chloroquine (1-1000 microM), and challenged with FITC-labeled rod outer segments for 24 hr. The phagocytized (bound and ingested) rod outer segments were measured fluorometrically, and the effect of the drugs on the phagocytosis was determined. The cytotoxicity of the drugs was evaluated by measuring their effects on mitochondrial enzyme activities (WST-1-test). The results showed that the test compounds inhibited the phagocytosis of rod outer segments in both D407 and pig retinal pigment epithelial cells. The phagocytic activity was more sensitive to tamoxifen (EC(50) 7.2 microM for D407 cells and 3.6 microM for pig retinal pigment epithelial cells) and toremifene (EC(50) 6.2 microM and 3.1 microM respectively) than to chloroquine (EC(50) 77.2 microM for D407 cells). The inhibition of rod outer segment phagocytosis in both cell cultures started at lower dose levels of test compounds than the cytotoxicity indicated by the WST-1-test. The experiments were carried out both in serum-free medium and serum-containing medium. Serum seemed to be a critical factor in the medium and caused difficulties in the interpretation of the results.

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