Overall performance of Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method with regard to solid and liquid chemicals testing.

A prospective study of the Bovine Corneal Opacity and Permeability (BCOP) Laser Light-Based Opacitometer (LLBO) test method was conducted to evaluate its usefulness to identify chemicals as inducing serious eye damage (Cat. 1) or chemicals not requiring classification for eye irritation (No Cat.) applying United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). The aim was to demonstrate the reproducibility of the BCOP LLBO protocol for liquids and solids and define its predictive capacity. Briefly, 145 chemicals were simultaneously tested with BCOP LLBO and OP-KIT (OECD TG 437), one to two times in one laboratory. When used to identify Cat. 1, the BCOP LLBO has a false negative rate (FNR) of 24.1% (N = 56) compared to 34.8% (N = 56) for the BCOP OP-KIT, with a comparable false positive rate (FPR, N = 89) of 18.5% and 20.8%, respectively. When used to identify chemicals not requiring classification (No Cat.) the BCOP LLBO and BCOP OP-KIT had a FNR (N = 104) of 6.2% and 7.2% and a FPR (N = 41) of 45.1% and 42.7%, respectively. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The OP-KIT and LLBO devices are interchangeable at no cost to data quality and reliability. The performance of the LLBO is at least as good as the OP-KIT, both methods can be used to identify UN GHS Cat. 1 and UN GHS No Cat. chemicals.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Development of a defined approach for eye irritation or serious eye damage for neat liquids based on cosmetics Europe analysis of in vitro RhCE and BCOP test methods.

The focus of Cosmetics Europe's ocular toxicity programme is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data and physicochemical properties. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. One key outcome of this project is that combining in vitro test methods (RhCE and BCOP LLBO) with physicochemical properties in a two-step Bottom-Up approach applicable to neat liquids, resulted in an improvement of the specificity, without reducing the sensitivity, when compared to the combination of in vitro methods alone. The Bottom-Up approach proposed here for neat liquids correctly predicted 58.3% (EpiOcular™ EIT followed by BCOP LLBO) to 62.6% (SkinEthic™ HCE EIT followed by BCOP LLBO) of No Cat., 59.1% to 68.7% of Cat. 2, and 76.5% of Cat. 1. Incorporating specific physicochemical properties with this Bottom-Up approach increased the correct identification of No Cat. neat liquids to between 72.7% and 79.7%.

Development of a defined approach for eye irritation or serious eye damage for liquids, neat and in dilution, based on cosmetics Europe analysis of in vitro STE and BCOP test methods.

The focus of Cosmetics Europe's programme on serious eye damage/eye irritation is on development of testing strategies and defined approaches for identification of ocular effects of chemicals in the context of OECD's Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye Damage and Eye Irritation. Cosmetics Europe created a comprehensive database of chemicals for which in vitro data are available with corresponding historical in vivo Draize eye data. This database allowed further exploration of the initially proposed strategies from the CON4EI project and to identify opportunities for refinement. The current analysis focused on the development of a defined approach, applicable to liquid non-surfactant chemicals, neat and in dilution, that can distinguish between the three UN GHS categories (Cat. 1, Cat. 2, and No Cat.). Combining the modified-protocol Short Time Exposure (STE) test method (OECD TG 491 with extension to highly volatile substances) with the Bovine Corneal Opacity and Permeability Laser Light-Based Opacitometer (BCOP LLBO) test method in a Bottom-Up approach identified 81.2% Cat. 1, 56.3% Cat. 2, and 85.3% No. Cat correctly, with an NPV of 96.7% and a PPV of 68.6%. Therefore, the performance of the defined approach was better than the standalone test methods.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Concomitant radiochemotherapy or accelerated radiotherapy: analysis of two randomized trials of the French Head and Neck Cancer Group (GORTEC).

In the early 1990s, when conventional radiotherapy (RT) was the standard of care in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), two main options were being tested to improve the efficacy and the therapeutic ratio of RT. The first approach evaluated the effect of adding chemotherapy (CT) simultaneously to RT (RT-CT), while the second approach assessed the effect of modified fractionated RT. To answer these two questions, in 1994, the French Group for Head and Neck Oncology Radiotherapy (GORTEC) initiated two randomized trials. A total of 494 patients were entered in these two parallel phase III multicenter trials comparing conventional RT (70 Gy in 35 fractions) either with concomitant RT-CT (226 patients; 70 Gy in 35 fractions with three cycles of a 4-day regimen comprising carboplatin and 5-fluorouracil [5FU]) or with very accelerated RT (268 patients) delivering 64 Gy in 3 weeks. The 5-year overall survival (OS), specific disease-free survival (DFS), and local-regional control rates were improved in favor of simultaneous RT-CT, whereas local-regional control was significantly improved with accelerated RT, along with a marginal effect on OS and DFS. This increased antitumor efficacy was in both cases associated with a marked increase in acute RT-induced toxicity, which was more pronounced with accelerated RT, whereas late effects were marginally increased with the addition of CT and not influenced by accelerated RT. We conclude that both concomitant RT-CT and accelerated RT improved tumor control rates, as compared to conventional RT, along with increased but manageable toxicity. The two regimens are currently being tested in an ongoing randomized study and also being compared to moderately accelerated RT and concomitant CT.

[Twin pregnancy in a unicornuate uterus with a rudimentary horn]. / Grossesse gémellaire et utérus unicorne avec corne rudimentaire communicante.

We describe the case of a rudimentary horn pregnancy coexistent with an intrauterine pregnancy. This situation is usually associated with rupture of the rudimentary horn and death of the correspondent twin. This is the first report on a multiple gestation with the two siblings successfully delivered by caesarean section in the two horns of a unicornuate uterus with rudimentary horn before any complication. Since the maternal mortality is higher in this situation, early ultrasound diagnosis is important to make the right decisions.

[Retained fecalith after laparoscopic appendicectomy]. / Coprolithe résiduel, après appendicectomie coelioscopique.

The subhepatic abscess due to retained fecalith is a rare complication following appendicectomy. The incidence of this complication is probably going to increase due to high rate of laparoscopic appendicectomy. We report 2 cases of subhepatic abscess 1 and 2 years after laparoscopic appendectomies. This potentially serious complication could be preventing with technical recommendations. When it occurs, this complication has to be directly treated by surgical drainage, percutaneous drainage couldn't be successful because it leaves fecalith in its place which is a cause of recurrence. Our reports are the first to use a laparoscopic treatment of this complication.

In situ gynogenetic haploid plants of chicory (Cichorium intybus L.) after intergeneric hybridization with Cicerbita alpina Walbr.

The possibility of obtaining haploid plants of chicory (Cichorium intybus L.) was investigated through intergeneric hybridization. Chicory plants (industrial chicory and Chioggia) were pollinated with pollen of Lactuca tatarica L. and Cicerbita alpina Walbr. Many achenes contained embryos which were rescued in vitro. Only a few embryos developed into plants which were then acclimatized in soil. Among them, three expressed a chicory phenorype and were haploid.

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum.

A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg(-1) H2O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca(2+) method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization.

Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10(6) mature pollen protoplasts were fused with 7·10(6) mesophyll protoplasts using a PEG/Ca(2+) method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l(-1)). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l(-1)). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Relationships among Cichorium species and related genera as determined by analysis of mitochondrial RFLPs.

Mitochondrial DNA polymorphism was employed to assess cytoplasmic diversity among cytoypes of the genus Cichorium and related genera of the tribe Lactuceae (Asteraceae). Hybridization patterns of total DNA using six restriction enzymes and five heterologous mtDNA probes were examined. From estimates of mtDNA diversity, Cichorium spinosum appeared as an ecotype of C. intybus rather than a separate species. Interspecific mtDNA polymorphism in the genus Cichorium was higher than that observed in Cicerbita Crepis, Lactuca and Tragopogon. Molecular data seemed to indicate that Catananche is very distant from the other genera examined. Intergeneric comparisons allowed the clustering of Cicerbita, Lactuca and Cichorium, genera which belong to different subtribes. However, further molecular investigations on a larger number of genera are needed to clarify the relationships among genera within and between subtribes of the tribe Lactuceae.