Complete Adjuvant Test Research Articles

Synthesis and Allergenic Potential of a 15-Hydroperoxyabietic Acid-like Model: Trapping of Radical Intermediates

To better understand the skin sensitization mechanism of 15-hydroperoxyabietic acid-like compounds, 2-(1‘-hydroperoxy-1‘-methylethyl)-4a-methyl-3,4,4a,5,6,7-hexahydronaphthalene 7 was synthesized. The allergenic activity of this compound was tested on mice using the local lymph node assay, and further evaluated on guinea pigs using the Freund's complete adjuvant test. Using these methods, hydroperoxide 7 was found to be a strong sensitizer. Radical-trapping experiments were carried out on this compound in the presence of the spin-trapping agent, 1,1,3,3-tetramethylisoindolin-2-yloxyl 17, using light to induce radicals. The formation of carbon-centered radicals derived from compound 7 by intramolecular cyclization of an oxygen-centered radical was observed. The reaction of these intermediates with 17 gave various adducts in a very low yield, but a 1/1 mixture of diastereomers 18a and 18b (0.1% yield) was isolated and characterized, together with compound 19 (0.03% yield). The results suggest that carbon-ce...

Dose-response studies of contact allergens using 3 guinea pigs models.

A multi-dose-response induction protocol for the guinea pig maximization test (GPMT), including a statistical computer program, has earlier been developed to improve the power of predictive tests for identification of contact allergens. This dose-response protocol, with 2 modifications (i.e., increased number of animals in each group and increased number of challenge concentrations) was evaluated in the GPMT, the cumulative contact enhancement test (CCET) and the Freund's complete adjuvant test (FCAT), using potassium dichromate and hydroxycitronellal as model contact allergens. Application of the dose-response protocol on the CCET and the FCAT resulted in either monotone or non-monotone curves with significant dose-response. However, application of the dose-response protocol on the GPMT gave curves with no significant dose-response. The protocol makes it possible to obtain an EC50 value, thus improving the possibility of ranking contact allergens, which is of substantial use for risk assessments. The dose-response protocol could benefit from a few adjustments: a wider span in the induction doses; change to simultaneous increase in intradermal and topical induction doses to obtain a proper dose-response for the GPMT; the addition of further challenge concentrations. In addition the computer program should allow calculation of threshold concentration for sensitization and EC50 value for a non-monotone curve.

Lack of antagonism to Ni2+ and Co2+ contact allergy from other essential divalent metal ions.

The potential antagonistic effects of Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ on contact allergy to Co2+ and Ni2+ were studied. The immune response was characterized by the Co2+ or Ni2+ mediated cellular [methyl-3H]thymidine uptake in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects contact-allergic to Co2+ and Ni2+ and 6 non-allergic control individuals. Results from the in vitro experiments were further evaluated with Co2+-sensitized guinea pigs according to the modified Freund's complete adjuvant test. Ni2+ and Co2+ (10-50 microM) significantly increased the lymphocyte proliferation in PBMC cultures from contact-allergic subjects in comparison with those from control individuals. Pretreatment of the PBMCs with Ca2+, Fe2+, Mg2+ (10-100 microM) or Mn2+ (1-10 microM) did not influence, while Zn2+ (100 microM) enhanced, and Cu2+ (5 and 10 microM) markedly reduced the Ni2+ and Co2+ mediated cellular [methyl-3H]thymidine uptake. The inhibition of the Ni2+- and Co2+-induced cell proliferation by Cu2+ in vitro was probably related to toxicity, since the viability of the cells was significantly reduced by applied combinations of Ni2+ or Co2+ with Cu2+. Topical pretreatment of Co2+-sensitized guinea pigs with maximum non-irritating doses of CuCl2 x 2H2O (0.8%) did not affect the challenge testing to CoCl2 x 6H2O (0.1 and 0.3%). In conclusion, our combined in vitro and in vivo results indicate that Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ are not able to antagonise the formation of Ni2+ and Co2+ antigens.

Skin sensitization to linalyl hydroperoxide: support for radical intermediates.

In order to better understand the skin sensitization mechanism of allylic hydroperoxides, linalyl hydroperoxide (1) and several of its potential rearrangement products-epoxylinalool (2), epoxynerol (3), epoxygeraniol (4), and furan (5) and pyran (6) derivatives-were synthesized. The sensitizing properties of these molecules have been screened on mice using the local lymph node assay (LLNA) and further evaluated on guinea pigs using the Freund's complete adjuvant test (FCAT). Linalyl hydroperoxide (1) and linalyl epoxide (2) were found to be sensitizers, while the other compounds were classified as mild sensitizers or nonsensitizers. In the guinea pigs, no cross-reactions were observed between skin sensitizers 1 and 2. Radical-trapping experiments were carried out on linalyl hydroperoxide (1) using TTBP as trapping agent and Fe(3+)-TPP as radical inducer. The major reaction taking place is the formation of a furan ring by intramolecular reaction of the oxygen-centered radical with the isoprenyl double bond with the formation of a tertiary radical. Reaction of this intermediate with radicals derived from TTBP gave compounds 10a,b in 25% yield. The second important reaction, accounting for 14%, is taking place on the allylic double bond with the formation of a less stable primary radical which is not trapped by a TTBP-derived radical but by a hydroxy radical to give a mixture of epoxides 3 and 4. These results are in favor of the formation of a carbon-centered reactive radical as intermediate in the skin sensitization to linalyl hydroperoxide.

Interactions of allergenic hydroperoxides with proteins: a radical mechanism?

1-(1-Hydroperoxy-1-methylethyl)cyclohexene was synthesized as a model compound for the study of the interaction of 15-hydroperoxyabietic acid-like terpenes with proteins. Two related epoxides, 1-(1-hydroxy-1-methylethyl)-2-oxabicyclo[4.1.0]heptane and 2,2-dimethyl-1-oxaspiro-[2.5]octan-4-ol, were also prepared as reference materials. Treatment of the hydroperoxide with FeCl3 and N alpha-Ac-Cys-OMe led to the formation of the corresponding alcohol and of both epoxides. The allergenic activity of these compounds was tested in guinea pigs using the Freund's complete adjuvant test. The hydroperoxide was found to be a strong sensitizer while both epoxides were found to be inactive at the same doses. The generation of highly-reactive radicals in the epidermis could lead to the formation of antigenic structures, the first step of the allergic contact dermatitis mechanism.

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 this study was to make further chemical analyses, to identify compounds formed by air exposure of d-limonene and to study their allergenic potential. d-limonene was found to be a sensitizer after prolonged exposure to air according to 2 Freund's complete adjuvant test (FCAT) experiments and 1 guinea pig maximization test (GPMT) study. No significant response was obtained to d-limonene not air exposed, even if the animals were sensitized to oxidized d-limonene. 5 main oxidation products of d-limonene were identified. (R)-(-)-carvone and a mixture of cis and trans isomers of (+)-limonene oxide were found to be potent sensitizers, while no significant reactions were obtained in the animals induced with a 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.

Experiences with Freund's complete adjuvant test (FCAT) when screening for contact allergens in colophony

A procedure, using Freund's complete adjuvant test (FCAT), for the determination of the allergenic potential of fractions and components in colophony of the gum rosin type is described and discussed. Gum rosin was shown to be a potent sensitizer in 11 test series (153 animals). FCAT is compared with the guinea pig maximization test (GPMT). Gum rosin was a potent sensitizer according to this method as well. The FCAT method was found to be advantageous over the GPMT method in that it is technically simpler to use and a smaller amount of test substance is needed. However, closed challenge was preferred to the prescribed open challenge. The importance of statistical evaluation of the results obtained in predictive testing is stressed.

The sensitizing potential of 2‐ethylhexyl acrylate in the guinea pig*

The sensitizing potential of 2-ethylhexyl acrylate in the guinea pig could be demonstrated by Freund's Complete Adjuvant Test. This acrylate ester is a common constituent of adhesive tape. Allergic reactions to several brands of adhesive tape were not observed in 2-ethylhexyl acrylate sensitized animals. Cross reactions with other acrylic monomers were observed.

Sensitizing potential of 12di(meth)acrylates in the guinea pig*

The aim of this study was to investigate the sensitizing potential of 12di(meth)acrylates, when tested in the Guinea Pig Maximization Test (GPMT) and Freund's Complete Adjuvant Test (FCAT). In these tests the same (molar) concentration was used for intradermal induction. The results (see table, below) show that dimethacrylates are moderate to strong sensitizers in the guinea pig. In the group of diacrylates, no sensitization to diethylene glycol diacrylate was observed. 1,2‐ethanediol diacrylate and 1,6‐hexanediol diacrylate sensitized a number of animals. The challenge reaction pattern of the FCAT with 1,4‐butanediol diacrylate, neopentane diol diacrylate and 1,5‐pentanediol diacrylate differed from that commonly observed with sensitizers. Positive reactions were seen only to the maximum non‐irritant concentration at 24 h in the challenge at day 21. At 48 h and in the second challenge at day 35, the reactivity had decreased or disappeared. This different reaction pattern makes it difficult to classify these monomers with certainty as sensitizers.In most of the FCATs, a decrease in reactivity in successive challenges was found. Possible explanations for this decrease and for the different challenge reaction patterns of some diacrylates are discussed.The skin irritant capacity of di(meth)acrylates was estimated after one open application. Diacrylates are very strong irritants on guinea pig skin. Dimethacrylates are weak irritants.

Sensitizing potential of 14 mono (meth) acrylates in the guinea pig*

The aim of this study was to investigate differences in the sensitizing potential of 14 mono(meth)acrylates, when tested by the guinea pig maximization test (GPMT) and Freund's complete adjuvant test (FCAT) with an identical, intradermal induction concentration. A new grading classification of the sensitization potential is proposed. Mono(meth)acrylates show a wide range of sensitizing potential. Compared with other (meth)acrylates, methyl methacrylate is a moderate sensitizer. Attention was paid to concomitant sensitization of additives. In 9 of 16 FCATs, concomitant sensitization occurred to the inhibitors hydroquinone and p-methoxyphenol.

Allergic contact dermatitis to costus: removal of haptens with polymers

Costus oil, an iol isolated from a Composite, Saussurea lappa C., used in perfumery, is responsible of numerous cases of allergic contact dermatitis (ACD). The haptens incriminated are sesquiterpene lactones, costunolide and dehydrocostuslactone. These lactones can be removed from the oil by using a polymer, aminoethyl-polystyrene, to which they become bound: the passing solution is lactone-free. A group of guinea-pigs could be successfully sensitized (Freund Complete Adjuvant Test) to costus essential oil, while another group, injected in the same way with polymer-treated essential oil (i.e. lactones) could not be sensitized. This polymer therefore provides a means to render complex plant extracts non-allergic.