Predicting skin sensitizer potency based on in vitro data from KeratinoSens and kinetic peptide binding: global versus domain-based assessment.

Three in vitro methods for the prediction of the skin sensitization hazard have been validated. However, predicting sensitizer potency is a key requirement for risk assessment. Here, we report a database of 312 chemicals tested in the KeratinoSens™ assay and for kinetic peptide binding. These data were used in multiple regression analysis against potency in the local lymph node assay (LLNA). The dataset covers the majority of chemicals from the validation of the LLNA to predict human potency and this subset was analyzed for prediction of human sensitization potency by in vitro data. Global analysis yields a regression of in vitro data to LLNA pEC3 with an R(2) of 60% predicting LLNA EC3 with a mean error of 3.5-fold. The highest weight in the regression has the reaction rate with peptides, followed by Nrf2-induction and cytotoxicity in KeratinoSens™. The correlation of chemicals tested positive in vitro with human data has an R(2) of 49%, which is similar to the correlation between LLNA and human data. Chemicals were then grouped into mechanistic domains based on experimentally observed peptide-adduct formation and predictions from the TIMES SS software. Predictions within these domains with a leave-one-out approach were more accurate, and for several mechanistic domains LLNA EC3 can be predicted with an error of 2- to 3-fold. However, prediction accuracy differs between domains and domain assignment cannot be made for all chemicals. Thus, this comprehensive analysis indicates that combining global and domain models to assess sensitizer potency may be a practical way forward.

Predicting the bioconcentration of fragrance ingredients by rainbow trout using measured rates of in vitro intrinsic clearance.

Bioaccumulation in aquatic species is a critical end point in the regulatory assessment of chemicals. Few measured fish bioconcentration factors (BCFs) are available for fragrance ingredients. Thus, predictive models are often used to estimate their BCFs. Because biotransformation can reduce chemical accumulation in fish, models using QSAR-estimated biotransformation rates have been developed. Alternatively, biotransformation can be measured by in vitro methods. In this study, biotransformation rates for nine fragrance ingredients were measured using trout liver S9 fractions and used as inputs to a recently refined in vitro-in vivo extrapolation (IVIVE) model. BCFs predicted by the model were then compared to (i) in vivo BCFs, (ii) BCFs predicted using QSAR-derived biotransformation rates, (iii) BCFs predicted without biotransformation, and (iv) BCFs predicted by a well-known regression model. For fragrance ingredients with relatively low (<4.7) log K(OW) values, all models predicted BCFs below a bioaccumulation threshold of 1000. For chemicals with higher (4.7-5.8) log K(OW) values, the model incorporating measured in vitro biotransformation rates and assuming no correction for potential binding effects on hepatic clearance provided the most accurate predictions of measured BCFs. This study demonstrates the value of integrating measured biotransformation rates for prediction of chemical bioaccumulation in fish.

Chemical reactivity and skin sensitization potential for benzaldehydes: can Schiff base formation explain everything?

Skin sensitizers chemically modify skin proteins rendering them immunogenic. Sensitizing chemicals have been divided into applicability domains according to their suspected reaction mechanism. The widely accepted Schiff base applicability domain covers aldehydes and ketones, and detailed structure-activity-modeling for this chemical group was presented. While Schiff base formation is the obvious reaction pathway for these chemicals, the in silico work was followed up by limited experimental work. It remains unclear whether hydrolytically labile Schiff bases can form sufficiently stable epitopes to trigger an immune response in the living organism with an excess of water being present. Here, we performed experimental studies on benzaldehydes of highly differing skin sensitization potential. Schiff base formation toward butylamine was evaluated in acetonitrile, and a detailed SAR study is presented. o-Hydroxybenzaldehydes such as salicylaldehyde and the oakmoss allergens atranol and chloratranol have a high propensity to form Schiff bases. The reactivity is highly reduced in p-hydroxy benzaldehydes such as the nonsensitizing vanillin with an intermediate reactivity for p-alkyl and p-methoxy-benzaldehydes. The work was followed up under more physiological conditions in the peptide reactivity assay with a lysine-containing heptapeptide. Under these conditions, Schiff base formation was only observable for the strong sensitizers atranol and chloratranol and for salicylaldehyde. Trapping experiments with NaBH3CN showed that Schiff base formation occurred under these conditions also for some less sensitizing aldehydes, but the reaction is not favored in the absence of in situ reduction. Surprisingly, the Schiff bases of some weaker sensitizers apparently may react further to form stable peptide adducts. These were identified as the amides between the lysine residues and the corresponding acids. Adduct formation was paralleled by oxidative deamination of the parent peptide at the lysine residue to form the peptide aldehyde. Our results explain the high sensitization potential of the oakmoss allergens by stable Schiff base formation and at the same time indicate a novel pathway for stable peptide-adduct formation and peptide modifications by aldehydes. The results thus may lead to a better understanding of the Schiff base applicability domain.

Chemical basis for the extreme skin sensitization potency of (E)-4-(ethoxymethylene)-2-phenyloxazol-5(4H)-one.

(E)-4-(ethoxymethylene)-2-phenyloxazol-5(4H)-one, commonly referred to as oxazolone, is the most potent skin sensitizer in published databases as determined with the murine local lymph node assay. It has been used very widely in immunological research to induce and elicit skin sensitization reactions in experimental animals. Nevertheless, no detailed study on the reactivity of oxazolone with proteins or peptides has been published, which would rationalize its unique sensitization potential from a chemical point of view. Peptide reactivity assays have been proposed as alternatives to animal tests to study the skin sensitization potential of test chemicals. Besides their application to reduce animal experimentation, peptide reactivity assays also allow one to gain mechanistic insights into the reactivity of test chemicals with proteins. In this case study, we applied different peptide reactivity assays to investigate and mechanistically rationalize the reactivity of oxazolone. Its sensitization potential could be linked to the following findings: (i) oxazolone reacts rapidly with the amine group in lysine with an addition-elimination reaction at the ethoxymethylene group to form stable products within minutes at physiological pH; (ii) sequentially different products with cysteine-peptides are formed, the most stable being an S-hippuryl-modification; and (iii) the S-hippuryl-modification can be shuttled to other nucleophilic sites; thus, also Lys residues can subsequently be modified with a hippuryl-moiety. This very rapid and diverse reactivity especially with lysine residues may explain why oxazolone forms sufficient stable novel epitopes on proteins to induce skin sensitization even at very low concentration.

Use of in vitro testing to identify an unexpected skin sensitizing impurity in a commercial product: a case study.

Due to regulatory constraints and ethical considerations, the quest for alternatives to animal testing has gained a new momentum. In general, animal welfare considerations and compliance with regulations are the key drivers for this research. Mechanistically based in vitro tests addressing specific toxicological questions can yield new information, for example on reactive components, and thus in certain cases the in vitro tests are not only second choice replacements of a 'gold standard' animal test but can also be used to develop safer products. Here we report a case study from the in vitro investigation on the commercial fragrance chemical Azurone. This compound was found to be a moderate skin sensitizer in the LLNA, whereas the structurally closely similar compound Calone is a non-sensitizer. A peptide reactivity assay indicated, that indeed Azurone yields peptide depletion, thus the in vitro assays confirmed the animal test result. LC-MS analysis of the peptide reactivity sample showed the presence of peptide adducts of unexpected molecular weight. They were consistent with the reaction of the peptide with a catechol related to Azurone. Detailed analytics indicated that indeed this catechol is present in the original batches as an impurity, but it has escaped quality control analysis, as it is not detectable in routine GC-analysis. A new purified batch was prepared, re-tested in the in vitro assays and predicted by the tests to be a non-sensitizer. A confirmatory LLNA test indeed yielded a significantly (10-fold) higher EC3 value of the new batch, but the LLNA was still positive. A dose-response study in the EpiSkin assay indicated that this molecule still has a significant skin irritation potential, which may generate the weak positive signal in the LLNA. This case study illustrates how the mechanistically based in vitro LC-MS peptide reactivity assay can be used to contribute to the understanding of the sensitization mechanism of a commercial product and help to define a safer product specification.

LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential.

A key step in the skin sensitization process is the formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. Based on this mechanistic understanding, there is a renewed interest in in vitro assays to determine the reactivity of chemicals toward peptides in order to predict their sensitization potential. A standardized peptide reactivity assay yielded a promising predictivity. This published assay is based on high-performance liquid chromatography with ultraviolet detection to quantify peptide depletion after incubation with test chemicals. We had observed that peptide depletion may be due to either adduct formation or peptide oxidation. Here we report a modified assay based on both liquid chromatography-mass spectrometry (LC-MS) analysis and detection of free thiol groups. This approach allows simultaneous determination of (1) peptide depletion, (2) peptide oxidation (dimerization), (3) adduct formation, and (4) thiol reactivity and thus generates a more detailed characterization of the reactivity of a molecule. Highly reactive molecules are further discriminated with a kinetic measure. The assay was validated on 80 chemicals. Peptide depletion could accurately be quantified both with LC-MS detection and depletion of thiol groups. The majority of the moderate/strong/extreme sensitizers formed detectable peptide adducts, but many sensitizers were also able to catalyze peptide oxidation. Whereas adduct formation was only observed for sensitizers, this oxidation reaction was also observed for two nonsensitizing fragrance aldehydes, indicating that peptide depletion might not always be regarded as sufficient evidence for rating a chemical as a sensitizer. Thus, this modified assay gives a more informed view of the peptide reactivity of chemicals to better predict their sensitization potential.

A specific bacterial aminoacylase cleaves odorant precursors secreted in the human axilla.

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.