An efficient method for the C-C bond formation via water soluble Na2PdCl4/sSPhos mediated Suzuki-Miyaura cross-coupling reaction of DNA-conjugated aryl iodide with (het)aryl boronic acids has been developed. This reaction proceeds at 37°C in water and acetonitrile (4:1) system. We also demonstrated that numerous aromatic and heteroaromatic boronic acids of different electronic natures, and harboring various functional groups, were highly compatible providing the desired coupling products in good to excellent yields. This DNA-compatible Suzuki-Miyaura cross-coupling reaction has strong potential to construct DNA-Encoded Libraries (DELs) in the context of drug discovery.

In vitro test batteries have become the standard approach to determine the genotoxic potential of substances of interest across industry sectors. While useful for hazard identification, standard in vitro genotoxicity assays in 2D cell cultures have limited capability to predict in vivo outcomes and may trigger unnecessary follow-up animal studies or the loss of promising substances where animal tests are prohibited or not desired. To address this problem, a team of regulatory, academia and industry scientists was established to develop and validate 3D in vitro human skin-based genotoxicity assays for use in testing substances with primarily topical exposure. Validation of the reconstructed human skin micronucleus (RSMN) assay in MatTek Epi-200™ skin models involved testing 43 coded chemicals selected by independent experts, in four US/European laboratories. The results were analysed by an independent statistician according to predefined criteria. The RSMN assay showed a reproducibly low background micronucleus frequency and exhibited sufficient capacity to metabolise pro-mutagens. The overall RSMN accuracy when compared to in vivo genotoxicity outcomes was 80%, with a sensitivity of 75% and a specificity of 84%, and the between- and within-laboratory reproducibility was 77 and 84%, respectively. A protocol involving a 72-h exposure showed increased sensitivity in detecting true positive chemicals compared to a 48-h exposure. An analysis of a test strategy using the RSMN assay as a follow-up test for substances positive in standard in vitro clastogenicity/aneugenicity assays and a reconstructed skin Comet assay for substances with positive results in standard gene mutation assays results in a sensitivity of 89%. Based on these results, the RSMN assay is considered sufficiently validated to establish it as a 'tier 2' assay for dermally exposed compounds and was recently accepted into the OECD's test guideline development program.

Measuring in vitro cytotoxicity is one method currently used to estimate damage to the eye after chemical exposure. The Cytosensor Microphysiometer method evaluates cytotoxicity by measuring the test material-induced reduction in the metabolic rate of L929 cells. Changes in metabolic rate are measured indirectly as a function of changes in the extracellular acidification rate of the cells. During exposure to increasing concentrations of a cytotoxic material, there is a decrease in the release of acid byproducts into the surrounding medium as the cells die. These acidic metabolic byproducts cause a measurable change in the pH of a lightly buffered medium, which can be measured by the Cytosensor Microphysiometer. The change in the pH of the medium over time is then converted into a metabolic rate estimate for the cells. The endpoint measurement from the assay is the metabolic rate decline of 50%, the MRD50 value (in units of mg/ml).

The in vitro human reconstructed skin micronucleus (RSMN) assay in EpiDerm (TM) is a promising new assay for evaluating genotoxicity of dermally applied chemicals. A global pre-validation project sponsored by the European Cosmetics Association (Cosmetics Europe - formerly known as COLIPA), and the European Center for Validation of Alternative Methods (ECVAM), is underway. Results to date demonstrate international inter-laboratory and inter-experimental reproducibility of the assay for chemicals that do not require metabolism [Aardema et al., Mutat. Res. 701 (2010) 123-131]. We have expanded these studies to investigate chemicals that do require metabolic activation: 4-n itroquinoline-N-oxide (4NQO), cyclophosphamide (CP), dimethylbenzanthracene (DMBA), dimethylnitrosamine (DMN), dibenzanthracene (DBA) and benzo(a)pyrene (BaP). In this study, the standard protocol of two applications over 48 h was compared with an extended protocol involving three applications over 72 h. Extending the treatment period to 72 h changed the result significantly only for 4NQO, which was negative in the standard 48 h dosing regimen, but positive with the 72 h treatment. DMBA and CP were positive in the standard 48 h assay (CP induced a more reproducible response with the 72h treatment) and BaP gave mixed results; DBA and DMN were negative in both the 48 h and the 72 h dosing regimens. While further work with chemicals that require metabolism is needed, it appears that the RMSN assay detects some chemicals that require metabolic activation (4 out of 6 chemicals were positive in one or both protocols). At this point in time, for general testing, the use of a longer treatment period in situations where the standard 48 h treatment is negative or questionable is recommended. (C) 2012 Elsevier B.V. All rights reserved.

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm™ for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm™ and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.

Recently, a novel in vitro reconstructed skin micronucleus (RSMN) assay incorporating the EpiDerm™ 3D human skin model (Curren et al., Mutat. Res. 607 (2006) 192–204; Mun et al., Mutat. Res. 673 (2009) 92–99) has been shown to produce comparable data when utilized in three different laboratories in the United States (Hu et al., Mutat. Res. 673 (2009) 100–108). As part of a project sponsored by the European cosmetics companies trade association (COLIPA), with a contribution from the European Center for the Validation of Alternative Methods (ECVAM), international prevalidation studies of the RSMN assay have been initiated. The assay was transferred and optimized in two laboratories in Europe, where dose-dependent, reproducibly positive results for mitomycin C and vinblastine sulfate were obtained. Further intra- and inter-laboratory reproducibility of the RSMN assay was established by testing three coded chemicals, N-ethyl-N-nitrosourea, cyclohexanone, and mitomycin C. All chemicals were correctly identified by all laboratories as either positive or negative. These results support the international inter-laboratory and inter-experimental reproducibility of the assay and reinforce the conclusion that the RSMN assay in the EpiDerm™ 3D human skin model is a valuable in vitro method for assessment of genotoxicity of dermally applied chemicals.

A novel in vitro human reconstructed skin micronucleus (RSMN) assay has been developed using the EpiDerm™ 3D human skin model [R. D. Curren, G. C. Mun, D. P. Gibson, and M. J. Aardema, Development of a method for assessing micronucleus induction in a 3D human skin model EpiDerm™, Mutat. Res. 607 (2006) 192–204]. The RSMN assay has potential use in genotoxicity assessments as a replacement for in vivo genotoxicity assays that will be banned starting in 2009 according to the EU 7th Amendment to the Cosmetics Directive. Utilizing EpiDerm™ tissues reconstructed with cells from four different donors, intralaboratory and interlaboratory reproducibility of the RSMN assay were examined. Seven chemicals were evaluated in three laboratories using a standard protocol. Each chemical was evaluated in at least two laboratories and in EpiDerm™ tissues from at least two different donors. Three model genotoxins, mitomycin C (MMC), vinblastine sulfate (VB) and methyl methanesulfonate (MMS) induced significant, dose-related increases in cytotoxicity and MN induction in EpiDerm™ tissues. Conversely, four dermal non-carcinogens, 4-nitrophenol (4-NP), trichloroethylene (TCE), 2-ethyl-1,3-hexanediol (EHD), and 1,2-epoxydodecane (EDD) were negative in the RSMN assay. Results between tissues reconstructed from different donors were comparable. These results indicate the RSMN assay using the EpiDerm™ 3D human skin model is a promising new in vitro genotoxicity assay that allows evaluation of chromosome damage following "in vivo-like" dermal exposures.

The cytosensor™ microphysiometer (μϕ) was investigated as a rapid, relatively inexpensive test to predict performance of skin cleansing wipes on the human 21-day cumulative irritation patch test (21CIPT). It indirectly measures metabolic rate changes in L929 cells as a function of test article dose, by measuring the acidification rate in a low-buffer medium. The dose producing a 50% reduction in metabolic rate (MRD50), relative to the baseline rate, is used as a measure of toxicity. The acute toxicity of the μϕ assay can be compared to the chronic toxicity of the 21CIPT, which is based largely on the exposure of test agents to the epidermal cells, resulting in damage and penetration of the stratum corneum leading to cell toxicity. Two series of surfactant-based cleansing wipe products were tested via the μϕ assay and 21CIPT. The first series, consisting of 20 products, was used to determine a prediction model. The second series of 38 products consisted of routine product development formulas or marketed products. Comparing the results from both tests, samples with an MRD50 greater than 50 mg/ml provided a 21CIPT score consistent with a product that performs satisfactorily in the market. When the MRD50 was greater than 78 mg/ml, the 21CIPT score was usually zero. The μϕ may be more sensitive than the 21CIPT for ranking minimally irritating materials. The μϕ assay is useful as a screen for predicting the performance of a wet wipes formula on the 21CIPT, and concurrently reduces the use of animals for safety testing in a product development program for cleansing wipes.

Assuring the safety of cosmetics and personal care products without testing in animals has long been the goal of many international companies. This concern has become even more important with the requirement of the Seventh Amendment to the Cosmetics Directive that after 2009 animal testing cannot be used to assess the eye or skin irritation potential of either cosmetics formulations or ingredients. To address this problem, the Alberto-Culver Company has developed a program to support the ocular safety evaluation of certain hair conditioners. This program relies on the results of a topical application of formulations to the surface of a three-dimensional, human cell-derived model of the corneal epithelium (EpiOcular, MatTek Corp., Ashland, MA, USA) and monitoring time to toxicity (ET50; MTT activity reduced to 50% of the control condition). Twenty-eight different formulations primarily based on either single or dual quaternary ammonium compound (quats) systems utilizing various combinations of seven different quats have been evaluated in the model. Potential safety of the materials was assessed by comparison to a benchmark material having a well established safety profile in commerce. Twenty-seven of the materials, including the benchmark, had ET50 values of 24 hours or greater, indicating that they were quite mild. The effectiveness of the system has been assessed by comparing the in vitro results with consumer experience information.

ALTEX 22, Special Issue 2005 183 The occurrence of asthma and Chronic Obstructive Pulmonary Disease (COPD) in industrialised countries has been increasing for the past 30 years. Local respiratory tolerance is a critical issue since lung irritation is a common acute side effect for inhaled drug. An in vitro airway epithelium model was implemented to determine irritancy potential of new chemical entities when given by the inhalation route to humans. The Calu-3 model consists of human bronchial epithelial cells of the airway tissue of the respiratory tract. Calu-3 cells when placed at the air-liquid interface form a 3-dimensional epithelium model, that develop tight junctions and functional barrier properties. It also induces ciliogenesis and produces mucus in our culture conditions. To determine the relevance and the reliability of this in vitro model, 8 chemicals intended for the treatment of COPD/asthma were tested in the Calu-3 model. A multiple endpoint analysis approach was used by measuring cell viability (MTT), inflammatory responses (interleukines and chemokines), tight junction disruption (trans-epithelial resistance) and production of mucin secretion on the apical side of the model. In vitro and in vivo animal data correlated suggesting that the Calu-3 model could be used to address lung irritancy potential of new chemical entities. Poster Pulmonary irritancy potential determination using an in vitro human airway epithelium model

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetic ingredients will not be allowed. Skin is one of the target areas of interest for many cosmetic products because it is generally the tissue with the highest exposure. Therefore we have begun development of a micronucleus assay using a commercially available 3-D engineered human skin model, EpiDermTM (MatTek Corp, Ashland, MA). We first evaluated whether a population of binucleated cells sufficient for a micronucleus assay could be obtained by exposing the tissue to 1-3 ug/ml cytochalasin B (Cyt B). The frequency of binucleated cells increased both with time and with increasing concentration of Cyt B. Cyt B at 3 ug/ml allowed us to reliably obtain 40–50% binucleated cells at 48 h and was used in future studies. The background frequency of micronuclei in this model is low (~0.1%) and reproducible. Studies with model genotoxins including mitomycin C, vinblastine sulfate and methylmethane sulfonate demonstrated that micronuclei can be reproducibly induced in this 3-D skin model. This is the first step in developing a routine “in vivo-like” assay for chromosomal damage in human tissue.

We have tested the irritancy of 10 materials of various chemical composition in three in vitro toxicity assays: the Skintex Dermal Assay system (In Vitro International, Irvine, CA, USA), the silicon microphysiometer (Molecular Devices Inc., Menlo Park, CA), and the Living Skin Equivalent (Organogenesis Inc., Cambridge, MA). The purpose was to discover to what degree the in vitro results predict in vivo skin irritation as seen in nine female volunteers over the course of a 5 day cumulative irritancy patch test. Two in vivo assessments of irritancy were made and compared to the in vitro results, a visual scoring system, and a potentially more objective bioengineering assessment: the chromameter. Measures of sensitivity, specificity, positive predictive value, and negative predictive value were used to compare in vitro with in vivo results. Comparison of results using correlation coefficients was avoided since statistically significant rank orders could not be demonstrated for either in vitro or in vivo data. For these in vitro systems, sensitivity ranged from 60 to 100%, specificity from 80 to 100%, positive predictive value from 75 to 100%, and negative predictive value from 67 to 100%. The assay that exhibited the best measures of sensitivity, specificity, and other parameters was the Living Skin Equivalent. This assay is also the one that most closely simulates human skin. The nonionic surfactant triton X-100 gave a false-positive result in both the Living Skin Equivalent and the silicon microphysiometer. False-positive results with nonionic surfactants are consistent with observations made in other cell-based in vitro systems. We caution that the results not be over-interpreted to imply superiority for any one system.

We have previously reported initial investigations of a novel micronucleus assay which utilizes a 3-dimensional human skin model EpiDerm™ supplied by MatTek Corporation (Ashland, MA). The advantage of this in vitro system is that it might potentially replace second-tier animal studies (or possibly first-tier non-specific traditional in vitro assays) in the genotoxicity evaluation of many materials, especially cosmetics ingredients. If so, it could then satisfy the requirement for non-animal testing of cosmetics ingredients after 2009 imposed by the European Seventh Amendment to the Cosmetics Directive. An important advantage of this assay system is that materials can be applied topically to the surface of the in vitro human skin model in a fashion similar to probable human exposure. In addition to previously reported positive responses with mitomycin C, vinblastine sulfate, MNNG and methyl methane sulfonate, four non-genotoxins (as shown by lack of micronucleus induction activity in an in vivo rodent skin model) have now been shown to induce no response in the reconstituted skin model. The non- genotoxins (4-nitrophenol, 1,2 epoxydodecane, trichloroethylene, and 2-ethyl-1,3-hexanediol) were tested up to levels of approximately 50% survival. The model has been further characterized by measuring expression of xenobiotic metabolizing enzymes. Although many enzymes were constitutively expressed, 3-methylcholanthrene and â-naphthoflavone strongly increased expression of CYP1A1 and CYP1B1, and slightly enhanced expression of CYP2C19, CYP2D6, CYP3A4 and CYP3A5. Overall, the in vitro model showed an enzyme expression pattern very similar to normal human skin. It appears that this model may be very useful for detecting of micronuclei induction in a structurally complex tissue of human origin.

The irritation potential of formulations and ingredients for industrial screening and product development is often conducted using in vitro 3-D human ocular and epidermal tissue constructs. To predict irritation potential after chemical exposure, tissue viability is typically determined by the ability of live cells to reduce MTT. Toxic exposures result in decreases in relative MTT reduction. However, two issues may contribute to inaccurate viability assessment: subtoxic exposures that induce higher metabolic rates typically greater than controls (i.e., hormesis) and chemicals that directly reduce MTT causing an overestimation of tissue viability (e.g., NaOH, α-tocopherol (α-t), ascorbic acid). For such chemicals, residues left on the tissues may increase the total MTT signal, so freeze-killed tissues are used to estimate chemical-mediated reduction of MTT. However, alternative methods of measuring tissue viability, such as amount of adenosine triphosphate (ATP) may be used. We compared these two methods by testing a series of model mild skin care formulations in 3-D human eye and skin constructs. The formulations were spiked with various concentrations of Triton ® to induce a range of toxic effects, and were prepared with and without α-t, a MTT reducer. For formulations with α-t, freeze-killed tissues were tested in parallel in both the MTT and ATP assays. The results showed the same irritancy predictions for the 4 formulations containing α-t as for the 4 control formulations without α-t (e.g., formula with highest Triton conc.: ET 50 eye = 172 and 157 min, ET 50 skin = 778 and 772 min, with and w/o α-t). The ATP assay provided the same rank order of irritancy as did the MTT assay although the relative viability values from the ATP assay at each exposure were overall lower (e.g., formula with highest Triton conc.: ET 50 eye = 14.5 and 12.9 min, ET 50 skin = 202 and 231 min, with and w/o α-t). In summary, the MTT assay of formulas capable of MTT reduction should include freeze-killed tissues, and the ATP assay can confirm the relative rank order of the irritancy predictions.

The bovine corneal opacity and permeability (BCOP) assay (Gautheron, 1992 & Sina, 1995), is used as an in vitro eye irritation screen for industrial hygiene, product development, and safety testing by measuring changes in corneal opacity, and permeability to fluorescein after chemical exposure. Histopathology has been used in BCOP studies to detect potential corneal injury, where the mode of chemical action might not induce opacity and permeability changes (Curren and Evans, 2000). Artifactual changes in the cornea associated with the collection, transportation, or BCOP methodology of the enucleated eyes have not been evaluated; therefore, corneas were excised and fixed in 10% buffered formalin at various steps in the assay process, paraffin embedded, H&E stained and evaluated using light microscopy. Stromal thickness and Descemet's Membrane (DM) thickness were measured along the entire length of the cornea. The epithelium, endothelium, and stroma were similar histologically among all groups. The normalized stromal thickness of the whole globe corneas (903.8 µm ± 122.9 µm), excised corneas immediately after enucleation (876.7 µm ± 84.2 µm), after the refrigerated transport (829.8 µm ± 63.4 µm), and at the end of the BCOP assay (721.2 µm ± 17.2 µm) suggest corneas undergo minimal artifactual changes as a result of refrigerted transport and the BCOP assay procedures.