Abstracts / Toxicology Letters 238S (2015) S56–S383 S243 to be positive in the Bhas 42 promotion assay. We have developed a suite of in vitro assays to allow us to compare the relative bio- logical effects of new categories of tobacco and nicotine delivery products with those of traditional cigarettes. The Bhas 42 promo- tion assay has been included in this test battery as it provides an in vitro surrogate for detecting tumour promoters. The activity of a tobacco heating product THP was compared to that of a reference combustible cigarette (3R4F) in the Bhas 42 promotion protocol. Six independent experiments were performed to assess reproducibil- ity of the responses to both products. Stock solutions of 30 mg/mL particulate matter (PM) were prepared from both products and frozen at -80 ◦ C until use. The products were smoked using the Health Canada Intense (HCI) regime. PM was tested up to a maxi- mum of 60 g/mL. Plates were scored and mean foci numbers were calculated for each treatment group. A clear concentration related response was obtained from the 3R4F PM, while the PM from the THP showed little activity and only gave a positive response in one experiment. The overall activity of PM from the THP was found not to differ significantly from a DMSO control at any concentration tested. 3R4F TPM induced significantly higher numbers of foci than control treatment at concentrations of 15 g/mL and above (Dun- nett’s test; p < 0.0001). A Generalised Linear Model with log-link function showed that the slopes of the responses across the concen- trations were significantly different between products (p < 0.0001). This study shows that the Bhas 42 promotion assay can distinguish between tobacco products, and could be included as part of a weight of evidence package for comparative risk assessment of tobacco and nicotine based products. http://dx.doi.org/10.1016/j.toxlet.2015.08.714 P11-025 Oral decitabine loaded PLGA nanoparticles combined with docetaxel suppress mammary carcinoma induced by DMBA in Sprague Dawley rats P. Jain 1,∗ , S.B. Mallik 2 , A.D. Chonkar 2 , M. Tiwari 1 , K. Nitesh 2 , J. Venkata Rao 1 , N. Udupa 2 1 Manipal University, Dept of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal, India 2 Manipal University, Manipal, India Decitabine (DEC) is an epigenetic drug. It has low oral bioavail- ability (4–5%) due to rapid degradation in acidic conditions and metabolism by cytidine deaminase in the liver and is therefore administered in clinical settings as i.v. infusion. In spite of the numerous advantages of oral chemotherapy, there is no conve- niently administrable oral dosage form available for DEC. Since PLGA has an advantage of overcoming acidic and enzymatic degra- dation, the present investigation was aimed at fabricating PLGA 50:50 nanoparticles of decitabine (DEC-NPs); and thereafter, exam- ine a combination treatment in vitro and in vivo with docetaxel (DTX). DEC-NPs were formulated by spontaneous emulsification solvent diffusion technique. The optimized formulation had PS of 124.3 ± 4.2 nm, ZP of -23.2 ± 1.2 mV, and EE of 41.8 ± 4.3%. A comparative study indicated that the cytotoxicity of DTX and DEC combination on MCF-7 cells was significantly higher (p < 0.05) than DTX and DEC alone. Furthermore, cell uptake studies in Caco-2 cells, evidenced enhanced uptake of DEC-NPs in comparison to DEC. For in vivo studies, 8week old female SD rats were divided into 7 groups (n = 6). Gp I was normal control, whereas groups II–VII were induced mammary tumor with 2 oral doses of DMBA in olive oil; 40 and 20 mg/kg. Gp II was kept as tumor c/c. Gp III received 7.5 mg/kg/week DTX i.p for 3 weeks; Gp IV received 33.7 mg/kg/week DEC i.p. for 3 weeks; Gp V was given same dose of DEC-NPs; Gp VI was given combination dose of Gp III and IV (DTX and DEC) while Gp VII was given combination dose of Gp III and V (DTX and DEC-NPs). The results demonstrated that the combina- tion of DEC and DEC-NPs with DTX significantly improved (p < 0.05) the tumor response rate, significantly reduced (p < 0.05) the tumor wt and volume when compared to single agents DTX or DEC. Treatment groups with DEC-NPs also displayed better response, as compared to DEC. The study proves that DEC and DEC-NPs enhance the anticancer potential of DTX. http://dx.doi.org/10.1016/j.toxlet.2015.08.715 P11-026 Characterisation of -catenin complexes by co-immunoprecipitation and high output Western blotting in different cell lines R.S. Haeussler 1,∗ , F. Treindl 1,2 , M. Schwarz 3 , M.F. Templin 1,2 1 NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany 2 Eberhard-Karls University Tuebingen, Pharmaceutical Biotechnology, Tuebingen, Germany 3 Eberhard-Karls University Tuebingen, Experimental and Clinical Pharmacology and Toxicology, Tuebingen, Germany Non-genotoxic carcinogens (NGCs) are substances, which lead to tumour formation without affecting the DNA directly. They often increase cell proliferation and have been shown to act as tumour promoters or receptor-mediators, which in succession can lead to the increased occurrence of tumours. Predicting the carcinogenic potential of these substances is challenging due to the observed variety in mode of action and to the obvious absence of geno- toxicity. Yet, different animal models have been developed, that allow mechanistic studies for certain classes of NGCs. In rodent liver, a single injection of diethylnitrosamine (DEN) followed by chronic treatment with the antiepileptic drug phenobarbital (PB) promotes the formation of hepatocellular tumours with activating mutations in Ctnnb1, encoding -catenin. This aberrant muta- tional activation in one of the central players of the canonical Wnt-pathway leads to the modulation of the signaling status of the pathway and changes central physiological processes such as cell proliferation, cellular metabolism, differentiation and apopto- sis. Since -catenin exerts its regulatory role as part of various protein–protein complexes, information on binding partners of - catenin in different complexes has proven to be helpful to gain a better understanding of the observed changes in cellular signal- ing. Therefore, we used the conventional co-immunoprecipitation to isolate protein complexes consisting of -catenin and differing interaction partners from mouse hepatoma cell lines prepared from tumours that carry different mutation e.g. in Ctnnb1 and p53. By coupling co-immunoprecipitation to a new, high output Western blotting approach (DIGI-West), a high resolution picture of the iso- lated -catenin complexes can be obtained. The approach allows the probing of a low amount of precipitate with hundreds of anti- bodies and thereby facilitates the identification of novel interaction partners of -catenin. The analyzed protein–protein complexes iso- lated from different cellular fractions (the soluble, cytosolic part and the chromatin-bound proteins) are presented and discussed in detail. http://dx.doi.org/10.1016/j.toxlet.2015.08.716