Genotoxicity testing using the micronucleus and comet assays in normal human cell based 3D epithelial models

Abstracts / Toxicology Letters 205S (2011) S60–S179

tude more potent than the formulations, SDS and BOS for capsase 3/7 activation in primary HUVEC cells. Conclusion: The R400 formulation and formulation blank had similar or lower toxicities compared with three common household detergents. Toxicity of R400 is unrelated to the active ingredient glyphosate. doi:10.1016/j.toxlet.2011.05.569

P1336 Delayed decontamination effectiveness following skin exposure to the chemical warfare agent VX D. Josse 1,∗ , G. Barrier 2 , C. Cruz 3 , M.C. Ferrante 3 , N. Berthelot 3 1

Crssa - Toxicologie, Institut de Recherche Biomédicale des Armées, La Tronche, France, 2 Sssm, SDIS06, Villeneuve Loubet Cedex, France, 3 Crssa Toxicologie, Institut de Recherche Biomédicale des Armées, La Tronche, France

Skin exposure to highly toxic chemicals such as the organophosphate nerve agent VX requires implementation of the decontamination process as quickly as possible. In a civilian context where a large number of individuals have been exposed to toxic chemicals, the victims must be moved to a decontamination station established by the fire department or set up at a hospital for patient thorough decontamination. The current French guidelines for decontamination in this context are: firstly, remove the outer layer of clothes, then apply the adsorbent Fuller’s Earth (FE) on exposed body surfaces, disrobe and have a shower. Objectives: (1) To determine the effectiveness of pre-showering decontamination procedures. (2) To evaluate the effect of substituting FE with another decontaminant and importance of the wash-in effect following skin showering. Methods: Skin decontamination, including showering, was performed in vitro by using split-thickness pig skin samples mounted on Franz-type diffusion cells. The following skin decontamination processes were performed: application of FE or RSDL® or Dakin® or baby wipes 30 min or 60 min post-exposure eventually followed with 1-min showering 60 min post-exposure; or 1-min showering 30 min or 60 min post-exposure to VX. The skin decontamination effectiveness was determined from the amount of VX that was absorbed in the skin and permeated through the skin 6 h postexposure. Results: 1 Pre-showering treatment with RSDL or Dakin 30 min postexposure to VX were the most effective decontamination procedures. 2 Skin decontamination effectiveness by FE was improved when followed with a shower. 3 There was no evidence for a wash-in effect. doi:10.1016/j.toxlet.2011.05.570

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P1337 Determination of airway genotoxicity potential using the EpiAirway in vitro human airway model and the comet assay H. Kandarova 1,∗ , A. Armento 2 , J. DeLuca 2 , Y. Kaluzhny 2 , M. Klausner 2 , P. Hayden 2 1

MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic, 2 MatTek Corporation, Ashland/MA, USA Purpose: Determination of genotoxicity potential is an important consideration for safety assessment of chemicals that may be inhaled during exposure to consumer products, occupational chemicals or environmental pollutants. Commonly used in vitro genotoxicity assays produce a high false positive rate, limiting their utility for predicting human genotoxicity. For assessment of organ-specific genotoxicity, 3D in vitro human tissue models with in vivo-like barrier function and metabolic capability will have improved biological relevance and predictive ability. Due to recently enacted legislation including REACH and a ban on animal testing of cosmetics by the 7th Amendment to the Cosmetics Directive, predictive organ specific genotoxicity tests are urgently needed. Methods: The EpiAirway model is produced from normal human airway epithelial cells and reproduces the 3D pseudostratified mucocilliary phenotype of in vivo proximal airways. The model has functional tight junctions and in vivo-like barrier properties as well as in vivo-like xenobiotic metabolizing capabilities. EpiAirway tissues were digested with (0.25%) trypsin to produce cell suspensions for Comet Assay experiments. 100 comets were visually scored per tissue in duplicate tissues. Visually scoring was conducted by assigning values for % tail DNA on a 5 category scale (0 being undamaged DNA to 4 > 80% DNA in tail). Results: Untreated control samples produced low background comet scores. Treatment of the EpiAirway tissues with prototypical genotoxins such as methyl methane sulfonate (MMS) prior to digestion produced statistically significant, dose-dependent increases in % tail DNA. The EpiAirway Comet Assay appears to be a promising approach to in vitro airway genotoxicity testing. doi:10.1016/j.toxlet.2011.05.571

P1338 Genotoxicity testing using the micronucleus and comet assays in normal human cell based 3D epithelial models Y. Kaluzhny 1 , P. Hayden 1 , H. Kandarova 2,∗ , S. Letasiova 2 , A. Armento 1 , J. DeLuca 1 , V. Karetsky 2 , M. Klausner 1 MatTek Corporation, Ashland, USA, 2 MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic

1

Safety assessment of new products for human use requires genotoxicity testing to ensure non-carcinogenicity. However, current in vitro assays have low specificity (resulting in a high rate of false positives) and in vivo genotoxicity testing was banned in 2009 by the 7th Amendment to the Cosmetics Directive. 3D human tissue models, which have in vivo-like barrier function and metabolism and which allow for topical exposure, are predicted to have improved biological relevance. The Reconstructed Skin Micronucleus (RSMN) and Comet assays (CA) that utilize MatTek’s highly differentiated EpiDermTM tissue model were adapted for use with tracheal, vaginal, oral, and corneal tissues. RSMN results showed statistically significant dose-dependent increases in cells containing micronuclei (MNC) for 9 direct genotoxins and 8 genotoxins that require metabolic activation, and no increases

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for 10 non-genotoxins. In addition, CA results showed statistically significant increases in % tail DNA after treatment with a model genotoxin. Utilizing the RSMN protocol with tracheal, vaginal, oral, and corneal tissue models, statistically significant increases in MNC (0.3–1.2%) were observed after treatment with genotoxins. Similarly, CA results with tracheal, vaginal, oral, and corneal tissue models showed increases in % tail DNA. Hence, the EpiDerm RSMN and CA assays can be applied to other in vitro human epithelial tissue models to predict genotoxic effects following real life exposure conditions. Together, RSMM and Comet assays will identify a wide spectrum of genotoxic hazards following exposure to consumer products, materials containing nanoparticles, occupational chemicals, or environmental pollutants. doi:10.1016/j.toxlet.2011.05.572

P1339 Zn2+ absorption in the different types of cells in the aquatic organism V.O. Khomenchuk ∗ , A.I. Gorda, O.I. Bodnar, V.Y. Byyak, V.V. Grubinko General Biology, Ternopil National Pedagogical University named after Volodymyr Gnatyk, Ternopil, Ukraine We studied the absorption of Zn2+ in vitro by isolated cells of the carp (gills, intestine, erythrocytes) and chlorella. The most Vmax were found in the enterocytes (18.0 nmol/g min), the minimum – in erythrocytes (4.6 nmol/g min). The cells of gills have the most affinity (Km ) to Zn2+ (Km = 2.12 ␮mol/l), the least – enterocytes (Km = 3.57 ␮mol/l). Vmax of absorption of Zn2+ by cells of chlorella decreases from 751.88 to 425.53 nmo1−1 during 6 h, and increases from 714.29 to 2857.14 nmo1/l from 12 to 72 h at concentrations of 1–5 mg/l. Km decreases from 4.76 to 0.727 mmol/l from 1 to 24 h of action. Membrane transport of Zn2+ is passive, enhances with the increase of concentration and time of the exposition. It is assumed that the transport of Zn2+ is performed by facilitated diffusion in the studied cells. The absorption of Zn2+ is determined by affinity zincbinding proteins, after saturation of sites-connecting the process becomes passive and uncontrolled by cell. The kinetic parameters of zinc absorption by cells of animal are less on the order for Vmax and on the two orders for Km of their values for chlorella. It is shows that animal’s proteins have higher affinity to the metal then cells of algae. doi:10.1016/j.toxlet.2011.05.573

P1340 The epiocularTM assay for testing eye irritation in vitro: In house validation with 60 test substance in a routine lab of the chemical industry A. Schrage 1 , S. Kolle 1,∗ , B. Wareing 1 , R. Tacou 1 , B. van Ravenzwaay 1 , H. Kandarova 2 , R. Landsiedel 3 1

Experimental Toxicology and Ecology, Ludwigshafen, Germany, MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic, 3 Environmental Toxicology Andecology, BASF SE, Ludwigshafen, Germany 2

and non-irritants (GHS category 2). Human reconstructed tissue models have been suggested for incorporation in a tiered test strategy to ultimately replace the Draize eye irritation test (OECD405). These models use cell death as an indicator of ocular irritation. We established and evaluated the EpiOcular assay to discriminate irritants from non-irritants. Test substances that decreased viability to ≤60% (compared to control) are considered eye irritants (GHS cat 1 or cat 2) and test substances with less effect are considered nonirritants. In addition to the EpiOcular Test we performed the BCOP assay (OECD437) and the direct peptide reactivity assay (DPRA, Gerberick). The tests were performed with 60 test substances including a broad variety of chemicals and formulations for which in vivo data (Draize test) were available: 18 severe irritants/corrosives (GHS category 1), 21 irritants (GHS category 2), and 21 non irritants. For the assessed data set the EpiOcular assay had a sensitivity of 90%, a specificity of 73% and an overall accuracy of 81%. Applying a lower viability threshold (50% instead of 60%) resulted in 82, 82 and 82%, respectively. The DPRA assay was not useful for prediction of eye irritation potential of the 60 compounds. Whereas the BCOP assay was – as expected – useful in further differentiating strong irritation effects. We will use the EpiOcular assay and the BCOP assay in a testing strategy to identify strong eye irritation, eye irritation and no eye irritation effects of test substances in routine testing of industrial chemicals and agrochemical formulations. doi:10.1016/j.toxlet.2011.05.574

P1341 Inhibition of ␤-tubulin ubiquitination by a Parkinson’s disease-related neurotoxic substance Y. Kotake ∗ , R. Kohta, Y. Hirokane, S. Ohta Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Substances that mimic the actions of causative gene products of familial Parkinson’s disease (PD) are candidate as causative agents of idiopathic PD. Recently, we reported that 1-benzyl1,2,3,4-tetrahydroisoquinoline (1BnTIQ), a PD-related neurotoxic substance, binds to beta-tubulin, which has been reported to be a substrate of parkin, a ubiquitin E3 ligase and a causative gene product of familial PD. Loss of function mutation of parkin is reported to result in loss of beta-tubulin ubiquitination. Therefore, we examined the effect of 1BnTIQ on ubiquitination of beta-tubulin. After SH-SY5Y cells were treated with 1BnTIQ, cells were lysed and collected for immunoprecipitation (IP)-Western blotting (WB) experiment. IP was performed using monoclonal beta-tubulin antibody, and WB was carried out using antibody against polyubiquitinated protein. In vitro ubiquitination assay was performed using a commercially available ubiquitination assay kit. The polyubiquitinated beta-tubulin level in human neuroblastoma SH-SY5Y cells was reduced in the presence of 10 ␮M 1BnTIQ for 48 h. It was reduced by 1 month exposure, even at concentrations (25–50 nM) as low as those detected in parkinsonian CSF. In vitro ubiquitination assay gave similar results. It is suggested that 1BnTIQ has the same effect on tubulin ubiquitination as does mutant parkin in familial PD. Taken together, substances which reduce polyubiquitination of tubulin such as 1BnTIQ are supposed to be candidates of etiological factors of PD. doi:10.1016/j.toxlet.2011.05.575

The bovine corneal opacity and permeability (BCOP) test was regulatorily accepted for the identification of corrosive and severe ocular irritants (GHS category 1) in 2009. But no in vitro test has been regulatorily accepted for the differentiation of ocular irritants