Sensitization, the prerequisite event in the development of allergic contact dermatitis, is a key parameter in both hazard and risk assessments. The pathways involved have recently been formally described in the OECD adverse outcome pathway (AOP) for skin sensitization. One single non-animal test method will not be sufficient to fully address this AOP and in many cases the use of a battery of tests will be necessary. A number of methods are now fully developed and validated. In order to facilitate acceptance of these methods by both the regulatory and scientific communities, results of the single test methods (DPRA, KeratinoSens, LuSens, h-CLAT, (m)MUSST) as well for a the simple '2 out of 3' ITS for 213 substances have been compiled and qualitatively compared to both animal and human data. The dataset was also used to define different mechanistic domains by probable protein-binding mechanisms. In general, the non-animal test methods exhibited good predictivities when compared to local lymph node assay (LLNA) data and even better predictivities when compared to human data. The '2 out of 3' prediction model achieved accuracies of 90% or 79% when compared to human or LLNA data, respectively and thereby even slightly exceeded that of the LLNA.

Human axillary odor is known to be formed upon the action of Corynebacteria sp. on odorless axilla secretions. The known axilla odor determinant 3-methyl-2-hexenoic acid was identified in hydrolyzed axilla secretions along with a chemically related compound, 3-hydroxy-3-methylhexanoic acid. The natural precursors of both these acids were purified from non-hydrolyzed axilla secretions. From liquid chromatography/ mass spectrometry analysis, it appeared that the acids are covalently linked to a glutamine residue in fresh axilla secretions, and the corresponding conjugates were synthesized for confirmation. Bacterial isolates obtained from the human axilla and belonging to the Corynebacteria were found to release the acids from these odorless precursors in vitro. A Zn 2؉ -dependent aminoacylase mediating this cleavage was purified from Corynebacterium striatum Ax20, and the corresponding gene agaA was cloned and heterologously expressed in Escherichia coli. The enzyme is highly specific for the glutamine residue but has a low specificity for the acyl part of the substrate. agaA is closely related to many genes coding for enzymes involved in the cleavage of N-terminal acyl and aryl substituents from amino acids. This is the first report of the structure elucidation of precursors for human body odorants and the isolation of the bacterial enzyme involved in their cleavage.The axilla region of humans contains a dense arrangement of apocrine, eccrine, and sebaceous glands, and it is an everyday experience, that volatile substances emanating from these areas make a key contribution to human body odor. Although this odor is perceived by today's society as mainly unpleasant, several studies indicate that it may contain chemical signals that affect the menstrual cycle (1) or that may be involved in a major histocompatibility complex allele-dependent mate selection (2). These studies point to an important role of body odors in the evolutionary history of man.Sweat as it is secreted by axillary glands is odorless. Since the pioneering work of Shelley et al. (3), it is known that (a) the typical strong axilla odor can only be released from apocrine secretions, and (b) that the action of skin bacteria is needed to generate the odoriferous compounds from non-smelling molecules present in these secretions. Indeed, the axilla is a skin region supporting a dense bacterial population, which is dominated by the two genera Staphylococcus and Corynebacteria (4, 5). Most individuals carry a flora that is dominated by either one of these two genera, and a strong correlation was found between a high population of Corynebacteria and a strong axillary odor formation (4, 6). As a practical consequence of these findings, halogenated antibacterials and aluminum preparations for reducing the bacterial population have become the main active ingredient of commercial deodorants for the last 40 years. The scientific conclusion from this early work was that axilla secretions contain non-odoriferous precursors that must be transformed by bacterial en...

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The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (PHL) is a major determinant in the biological control of a range of plant pathogens by many fluorescent Pseudomonas spp. A 4.8-kb chromosomal DNA region from Pseudomonas fluorescens Q2-87, carrying PHL biosynthetic genes, was used as a probe to determine if the PHL biosynthetic locus is conserved within PHL-producing Pseudomonas strains of worldwide origin. The phl gene probe hybridized with the genomic DNA of all 45 PHL-producing Pseudomonas strains tested, including well-characterized biocontrol strains from the United States and Europe and strains isolated from diseasesuppressive soils from Switzerland, Washington, Italy, and Ghana. The PHL producers displayed considerable phenotypic and genotypic diversity. Two phenotypically distinct groups were detected. The first produced PHL, pyoluteorin, and hydrogen cyanide and consisted of 13 strains from almost all locations sampled in the United States, Europe, and Africa. The second produced only PHL and HCN and consisted of 32 strains from the U.S. and European soils. Analysis of restriction patterns of genomic DNA obtained after hybridization with the phl gene probe and cluster analysis of restriction patterns of amplified DNA coding for 16S rRNA (ARDRA) and randomly amplified polymorphic DNA (RAPD) markers indicated that the strains that produced both PHL and pyoluteorin were genetically highly similar. In contrast, there was more diversity at the genotypic level in the strains that produced PHL but not pyoluteorin. ARDRA analysis of these strains indicated two clusters which, on the basis of RAPD analysis, split into several subgroups with additional polymorphisms. In general, the occurrence of phenotypically and genotypically similar groups of PHL producers did not correlate with the geographic origin of the isolates, and highly similar strains could be isolated from diverse locations worldwide.

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h-Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSens(TM) assay which is based on detection of Nrf2-induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data-driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937- test, the DPRA and KeratinoSens(TM) . It can serve to develop data-driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read-across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three-fold smaller datasets. Analysis of the dose-response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false-negative and false-positive in two tests helped to define limitations in the tests but also in the database derived from animal studies.