Ulrika Nilsson

Air oxidation of d‐limonene (the citrus solvent) creates potent allergens

Products containing as much as 95% of d‐limonene are used for, e.g., degreasing metal before industrial painting and for cleaning assemblies. Experimental studies on the sensitizing potential of limonene show diverging results. In a previous study, we found that the sensitizing potential of d‐limonene increased with prolonged air exposure. The aim of 1 his study was to make further chemical analyses, to identify compounds formed by air exposure of d‐limonende and to study their allergenic potential. d‐limonene was found lo be a sensitizer after prolonged exposure to air according to 2 Freunds complete adjuvant test (FCAT) experiments and I guinea pig maximization test (GPMT) study. No significant response was obtained to d‐limonene air exposed, even if the animals were sensitized to oxidized d‐limonene. 5 main oxidation products of d‐limoncne were identified. (R)‐(–)‐carvone and a mixture of cis and trans isomers of (+)‐limonene oxide were found to he potent sensitizers, while no significant reactions were obtained m the animals induced with 8 mixture of cis and trans isomers of (–)‐carveol. It can be concluded that air oxidation of d‐limonene is essential for its sensitizing potential, and that potent allergens are created.

Analysis of contact allergenic compounds in oxidizedd-limonene

SummaryA detailed chemical analysis and quantification of oxygen-containing monoterpenes in auto-oxidizedd-limonene was performed. Some of these, e.g. the cis- and trans-isomers of limonene-2-hydroperoxide, have previously been shown to exhibit strong contact allergenic properties. GC-MS with chemical ionization in negative ion mode was shown to be a successful method for the identification and determination of the molecular weight of chemically unstable limonene hydroperoxides. An HPLC method for isolation of individual compounds in auto-oxidizedd-limonene is presented. Two different stationary phases were used in normal phase mode, cyanopropyl- and diamino-modified silica. The method described can be used for the isolation of individual contact allergens in sensitization experiment vehicles, such as petrolatum and olive oil. This makes it possible to study whether test compounds, such as hydroperoxides, are chemically stable during a sensitization experiment.

15-hydroperoxydehydroabietic acid—a contact allergen in colophony from Pinus species

A new hydroperoxide, 15-hydroperoxydehydroabietic acid (15-HPDA), with contact allergenic properties has been detected in rosin obtained from Pinus species. Detection was facilitated using a synthetic preparation of 15-HPDA for reference purposes. The synthesis and the detection (HPLC and GC) of 15-HPDA in rosin are described. The allergenic activity of 15-HPDA was studied in an experimental sensitization test on guinea-pigs.

The allergenicity of glycerol esters and other esters of rosin (colophony)

To investigate whether esterification of rosin with polyalcohols changes its allergenic potential. abietic acid, the main component of rosin, was esterified with glycerol at high temperature. The major product formed was isolated and identified, using nuclear magnetic resonance (NMR). infra‐red IR) and mass spectrometry (MS) analyses, as glyceryl triabietate (GTA), an ester between one glycerol molecule and 3 abietic acid molecules. According to animal experiments GTA was not allergenic and no cross‐reactivity was seen to allergens in unmodified rosin, when testing patients allergic to unmodified rosin, no reactions were found to GTA. Some of the patients reacted to glycerol‐ and pentaerythritol‐esterified rosins. According to HPLC analyses, these esterified rosins still contained unmodified material to which the patients may have reacted. It seems that the esterification of rosin with poly alcohols such as glycerol reduces its allergenic activity, possibly because of the formation of much larger molecules with reduced bioavailability. However, making methyl esters of rosin causes little alteration in the molecular weights of the components and. when unmodified and methylated rosin were tested in patients, we saw no difference between the 2 forms.

Rosin allergy: identification of a dehydroabietic acid peroxide with allergenic properties

A peroxide of dehydroabietic acid was isolated from rosin using flash chromatography and preparative HPLC. It was identified by 1NMR and MS. In animal experiments, this peroxide cross-reacted with a previously identified allergen in rosin, 15-hydroperoxyabietic acid (15-HPA), despite differences in molecular weight and unsaturation. Both substances are able to react via a radical mechanism generating structurally similar molecules. In patch testing of patients, no reactions were observed to the peroxide. Low skin penetration of the peroxide could be the explanation for this. The peroxide seems of little clinical importance. The observed cross-reactivity is an indication of antigen generation via a radical mechanism. Only a few compounds that react with radical mechanisms to form antigens are described in the literature.

Allergenicity of rosin (colophony) esters: (II). Glyceryl monoabietate identified as contact allergen

In the esterification of rosin with glycerol, the main compound formed, glyceryl triabietate, shows low allergenic activity. In this study, compounds formed in smaller amounts, when abietic acid (main component in rosin) was esterified with glycerol, were identified as glyceryl‐1‐monoabietate (GMA), glyceryl‐1,2‐diabietate (GDA1,2) and glyceryl‐1,3‐diabietate (GDA1,3), using nuclear magnetic resonance (NMR), infra‐red (IR) and mass spectrometry (MS) analyses. According to animal experiments, GMA was a contact allergen. No cross‐reactivity was seen to allergens in unmodified rosin. Some patients allergic to unmodified rosin reacted when tested with GMA. No reactions were seen to the 2 diabietates. Some patients also reacted to commercial glycerol‐modified rosins. GMA together with unmodified abietic acid were identified in these rosin samples. The reactions seen in rosin‐sensitive patients to commercial glycerol‐esterified rosins probably derive from the unmodified material still present in the product, but could also be the result of GMA obtained from the glycerol derivatization.

Formation of chlorinated polycyclic aromatic hydrocarbons in different chlorination reactions

Abstract Eleven of the most commonly found PAHs were subjected to different chlorination reactions, the products formed being then analyzed by GC/MS. These reactions were performed in carbon tetrachloride solution and on carbon black or urban dust particles, and the influence of UV-irradiation on the formation of chlorinated products was studied. When dissolved in carbon tetrachloride and irradiated with UV-light, several chloro-added PAHs were formed, of which some have shown direct mutagenic effects in the Ames assay. A large number of isomers of chloro-substituted PAHs, mainly mono- and dichloro-derivatives, was also detected. Some of these were shown to be degradation products of the chloro-added compounds. UV-irradiation had a minor effect on the formation of chlorinated products, when the PAHs were adsorbed and chlorinated on carbon black or on urban dust particles. The chlorinated products were similar to those isomers formed when the solution of PAHs was chlorinated in the absence of light, i.e. mainly products originating from electrophilic aromatic substitution. The stabilities of chloro-PAHs towards photochemical degradation were shown to increase when adsorbed on soot particles. An increasing number of chlorosubstituents on the aromatic nuclei also enhanced the stabilities of the PAHs examined.

LETHALITY AND EROD-INDUCING POTENCY OF CHLORINATED CHRYSENE IN CHICK-EMBRYOS

Chrysene was non-specifically chlorinated and the toxic potency of the mixture formed was studied. The chlorinated mixture was considerably more potent than the parent compound in terms of embryolethality and EROD and AHH induction in a 2-week test in chick embryos. The chlorinated chrysene caused anomalies, including edema and beak defects, similar to those previously found after treatment of chick embryos with coplanar PCBs. Chlorinated chrysene was also much more potent than chrysene as an inducer of EROD in a 72-hour test in chick embryos in ovo and in chick embryo liver in vitro. It seems that the mechanism of toxicity of chlorinated chrysene in chick embryos is similar to that of the coplanar PCBs and other Ah receptor ligands. The effects of the chlorinated mixture were mainly accounted for by 6- chlorochrysene and 6,12-dichlorochrysene.