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Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch), Several aromatic aldehydes such as 3-(4-tert-butylphenyl)-2-methylpropanal were shown to adversely affect the reproductive system in male rats following oral gavage dose of ≥ 25 mg/kg bw/d. It was hypothesized that these aldehydes are metabolized to benzoic acids such as p-tert-butylbenzoic acid as key toxic principle and that Coenzyme A (CoA) conjugates may be formed from such acids. Here we performed a detailed structure activity relationship study on the formation of benzoic acids from p-alkyl-phenylpropanals and related chemicals in rat hepatocytes in suspension. Formation of CoA conjugates from either p-alkyl-phenylpropanals directly or from their benzoic acid metabolites was further assessed in plated rat hepatocytes using high resolution LC-MS. All of the test chemicals causing reproductive adverse effects in male rats formed p-alkyl-benzoic acids in rat hepatocytes in suspension. Compounds metabolized to p-alkyl-benzoic acids led to accumulation of p-alkyl-benzoyl-CoA conjugates at high and steady levels in plated rat hepatocytes, whereas CoA conjugates of most other xenobiotic acids were only transiently detected in this in vitro system. The correlation between this metabolic fate and the toxic outcome may indicate that accumulation of the alkyl-benzoyl-CoA conjugates in testicular cells could impair male reproduction by adversely affecting CoA-dependent processes required for spermatogenesis. This hypothesis prompted a search for new p-alkyl-phenylpropanal derivatives which do not form benzoic acid metabolites and the corresponding CoA conjugates. It was found that such metabolism did not occur with a derivative containing an o-methyl substituent, ie, 3-(4-isobutyl-2-methylphenyl)propanal. This congener preserved the fragrance quality but lacked the male reproductive toxicity in a 28-day rat study, as predicted from its in vitro metabolism.

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.

Upon prolonged exposure to air, limonene is prone to oxidation. Hydroperoxides formed upon oxidation are strong skin sensitizers in animal tests and give positive patch tests in human dermatitis patients. However, there is no analytical evidence indicating potential sources from which the public can be exposed to sufficient quantities of limonene hydroperoxide for induction of skin sensitization to occur. It has been proposed that fragranced products might lead to such exposure. Here, we developed analytical methods using mass spectrometry (MS) in combination with gas chromatography (GC)-MS and liquid chromatography (LC)-MS to detect limonene-2-OOH. GC-MS has a low sensitivity, whereas LC-MS was found to be a sensitive method to detect this hydroperoxide. However, selectivity of LC-MS is low in complex fine fragrances owing to limited separation and unspecific ions. We developed an additional method based on selective reduction of limonene-2-OOH to carveol by triphenylphosphine (PPh3). Quantitative carveol formation from the hydroperoxide in the presence of PPh3 could be verified in hydroperoxide-spiked fragrances. These methods were then applied to stability studies. We found a high stability of limonene in eaux de toilette and the PPh3-reduction assay gave no indication of hydroperoxide formation in repeatedly opened, half-empty bottles stored for 9 months. Aged fine fragrances retrieved from consumers contained an average of 1990 µg/g limonene, but only traces of carveol were formed in a minority of the samples in the presence of PPh3. Control samples were spiked with trans-limonene-2-OOH. Quantitative trans-carveol formation in the presence of PPh3 indicated that the method would detect the hydroperoxide in these consumer products if it was present at higher concentrations. In conclusion, limonene-2-OOH may be detected in complex fragrance samples by selective reduction followed by GC-MS. However, we could not find evidence for significant limonene-2-OOH formati

Hydroperoxides can act as specific haptens and oxidatively modify proteins. Terpene hydroperoxides trigger unusually high frequencies of positive skin reactions in human patients if tested at high concentrations. It is unknown whether this is due to specific hapten formation. Here, we show that both terpene hydroperoxides and the endogenous hydroperoxide formed from squalene can oxidatively modify tryptophan. Oxidative modifications of Trp were recently postulated to explain cross-sensitization between unrelated photosensitizers. Current observations may extend this hypothesis: Oxidative events triggered by endogenous hydroperoxides and hydroperoxides/oxidants derived from xenobiotics might lead to a sensitized state detected by patch tests with high concentrations of hydroperoxides.