Me-too validation study for in vitro skin irritation test with a reconstructed human epidermis model, KeraSkin™ for OECD test guideline 439.

We conducted a me-too validation study to confirm the reproducibility, reliability, and predictive capacity of KeraSkin™ skin irritation test (SIT) as a me-too method of OECD TG 439. With 20 reference chemicals, within-laboratory reproducibility (WLR) of KeraSkin™ SIT in the decision of irritant or non-irritant was 100%, 100%, and 95% while between-laboratory reproducibility (BLR) was 100%, which met the criteria of performance standard (PS, WLR≥90%, BLR≥80%). WLR and BLR were further confirmed with intra-class correlation (ICC, coefficients >0.950). WLR and BLR in raw data (viability) were also shown with a scatter plot and Bland-Altman plot. Comparison with existing VRMs with Bland-Altman plot, ICC and kappa statistics confirmed the compatibility of KeraSkin™ SIT with OECD TG 439. The predictive capacity of KeraSkin™ SIT was estimated with 20 reference chemicals (the sensitivity of 98.9%, the specificity of 70%, and the accuracy of 84.4%) and additional 46 chemicals (for 66 chemicals [20 + 46 chemicals, the sensitivity, specificity and accuracy: 95.2%, 82.2% and 86.4%]). The receiver operating characteristic (ROC) analysis suggested a potential improvement of the predictive capacity, especially sensitivity, when changing cut-off (50% → 60-75%). Collectively, the me-too validation study demonstrated that KeraSkin™ SIT can be a new me-too method for OECD TG 439.

Assessment of skin sensitizing potential of metals with ß-galactosidase-expressing E. coli culture system.

It has been a challenge to develop in vitro alternative test methods for accurate prediction of metallic products which may exert skin sensitization, as several test methods adopted by OECD were relatively ineffective in assessing the capacity for metallic compounds to exert sensitizing reactions, compared with organic test substances. Based upon these findings, a system that incorporates ß-galactosidase producing E. coli cultures was tested for its predictive capacity to well-known metallic sensitizers. In this system, E. coli cells were incubated with metal salts at various concentrations and ß-galactosidase suppression by each test metal was determined. Fourteen local lymph node assay (LLNA) categorized metal salts were examined. Although color interference from metal salts was minimal, a fluorometric detection system was also employed using 4-methylumbelliferyl galactopyranoside as a substrate for ß-galactosidase to avoid the color interference, concomitantly with the original UV-spectrometric method. Data demonstrated that two detection methods were comparable and complementary. In addition, most of the metallic sensitizers were correctly identified at 0.6 and 0.8 mM concentrations. Despite the lower specificity obtained in the current study and small number of substances tested, the developed method appears to be a relatively simple and effective in vitro method for detecting metallic sensitizers. When 61 chemicals tested in the ß-galactosidase producing E. coli cultures including the present study were collectively analyzed, the prediction capacity was as high as other OECD-adopted tests: 95.6% of sensitivity, 66.7% of specificity, and 88.5% of accuracy. It is important to emphasize that animals or mammalian cell cultures were not required in the current method, which are in accordance with the EU guidelines on restricted or banned animal testing.

Risk assessment of tetrabromobisphenol A on cyclooxygenase-2 expression via MAP kinase/NF-kappaB/AP-1 signaling pathways in murine macrophages.

Tetrabromobisphenol A [2,2-bis-(3,5-dibromo-4-hydroxyphenyl)propane; TBBPA] is used worldwide as a flame retardant in numerous products. In the present study, the effects of TBBPA were examined on the expression of cyclooxygenase-2 (COX-2), inflammation-related cytokines, transcription factors, and signaling pathways responsible for transcriptional activation of the COX-2 gene in murine RAW 264.7 macrophages. Exposure to TBBPA markedly enhanced the production of prostaglandin E(2), a major COX-2 metabolite, in macrophages. TBBPA concentration-dependently increased the levels of COX-2 protein and mRNA. In addition, TBBPA increased the secretion and mRNA levels of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta. Transfection of a human COX-2 promoter construct demonstrated that TBBPA induced COX-2 promoter activity. Furthermore, transfection with pNF-kappaB-Luc and pAP-1-Luc plasmid revealed that TBBPA activated the NF-kappaB and AP-1 sites. Phosphatidylinositol 3 (PI3) kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by TBBPA. Our data demonstrate TBBPA-induced COX-2 and proinflammatory cytokine expression occurs through the PI3-kinase/Akt/MAP kinase/NF-kappaB/AP-1 pathways.

Nephrotoxic potential and toxicokinetics of tetrabromobisphenol A in rat for risk assessment.

Tetrabromobisphenol A (TBBPA), one of the most widely used global brominated flame retardants, is used to improve fire safety of laminates in electrical and electronic equipment. To investigate the nephrotoxic potential of TBBPA and its toxicokinetic profile in rats, single-dose and daily 14-d repeated-dose toxicity studies at 200, 500, or 1000 mg/kg were performed. Several biochemical parameters were analyzed to evaluate nephrotoxicity of TBBPA. High-dose 1000 mg/kg TBBPA significantly elevated renal thiobarbituric acid-reactive substance (TBARS) levels, and superoxide dismutase (SOD) activity was increased at all 3 doses administered. This was associated with no change in the activity of catalase (CAT). Our results suggest that acute 1-d high-dose administration of TBBPA produced transient renal changes at 5 h. Subsequently, TBBPA in serum, urine, and kidney was determined by liquid chromatography-mass spectroscopy (LC/MS). Toxicokinetic studies indicated that TBBPA shows relatively a short half-life (7-9 h) and was eliminated almost completely in feces by 2 d. Based on the results from the 14-d repeated-dose study, TBBPA did not accumulate in the rat, and was eliminated in feces. The present results suggested that TBBPA may not be toxic to kidney, as the chemical is not bioavailable and is not present in renal tissue.