Evaluation of contact allergy to chemicals using laser Doppler flowmetry (LDF) technique

Skin blood flow in allergic contact reactions and cross-sensitivity were evaluated using laser Doppler flowmetry (LDF) to study the dose-response relationships in phases of induction and challenge in guinea pigs. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with different doses of DNCB and 2,4-dinitrobenzene sulfonic sodium salt (DNBS). The skin reactions were evaluated by LDF and visual reading score. The results indicated that there were dose-response relationships between the doses of DNCB and LDF measurements in both phases of induction and challenge, that there was a cross-reaction between DNCB and DNBS, and that the reactions at 24 h were greater than that at 48 h after removal of the patches. LDF may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. This is because vascular dilatation and increase of flow already reaches a maximum in weak reactions. The more advanced phases are dominated by oedema formation. This is simply the nature of the inflammatory reaction, rather than a methodological error. The important point is that LDF can separate positive reactions from negative/uncertain reactions. The results indicated that LDF, as a non-invasive technique, may objectively and quantitatively evaluate the dose-response relationships of contact sensitivity of sensitizers.     

Topical application of artesunate on guinea pig allergic contact dermatitis

Artesunate is derived from the Chinese medicinal herb qinghao. Many animal studies suggest that systemic artesunate has immunoregulatory activity. We investigated the effect of topical artesunate on contact sensitivity in guinea pigs sensitized with DNCB. Female hairless guinea pigs were used and the contact reaction graded by visual (5 point) score and erythema measured with a color analyzer. Topical artesunate inhibited the elicitation reaction of contact hypersensitivity when given at the 1st and 12th h after challenge ( p < 0.05), but showed no effect when given from day 3 to day I before challenge ( p > 0.05). Artesunate had no effect on toxic (irritant) contact dermatitis from 20% croton oil ( p > 0.05). The results indicate that topical application of artesunate may offer a novel treatment of allergic contact dermatitis and other cutaneous immune-mediated disorders.     

Predictive evaluation in animals of the contact allergenic potential of medically important substances I. Comparison of different methods of inducing and measuring cutaneous sensitization

Groups of guinea pigs were sensitized with a 0.1% solution of dinitrochlorobenzene (DNCB) by the Draize intracutaneous method, The course of the induction process, the influence of the vehicles used and the extent to which the reactions are amenable to assessment according to objective criteria were examined. The sensitivity of the standardized Draize test was then compared with that of various other sensitization techniques, including:
The intracutaneous test with adjuvant (optimization test)
The maximization test according to Magnusson & Kligman (1969)
The epidermal sensitization test
The epidermal sensitization lest wish prior irritation of the contact site (by croton oil or sodium lauryl sulphate).
Comparison of these methods revealed that either the additional application of adjuvant or prior irritation of the contact site augmented the degree of sensitization to DNCR just as greatly as the simultaneous use of adjuvant and prior irritation of the skin, (maximization test.). The improved sensitization methods, and in particular the standardised optimization test, may prove to be of particular value for the study of so-called weak allergens.     

Experimental study of the potential for contact sensitization and cross-reaction of imidazole antifungals

We examined the potential for contact sensitization of miconazole nitrate and croconazole hydrochloride and the cross-reaction between them in guinea pigs by the maximization test of Magnusson and Kligman. Contact sensitivity was induced by croconazole hydrochloride in 5 out of 7 animals which, after being injected with 5% croconazole hydrochloride, underwent a closed patch with 25% croconazole hydrochloride. Contact sensitivity was not induced by miconazole nitrate. The 5 animals sensitized to croconazole hydrochloride were tested with 8 other imidazole antifungals and positive reactions were observed to oxiconazole nitrate in 2 of the 5 animals. This response may be a cross-reaction.     

Influence of area of application of allergen on sensitization in contact dermatitis

There are at least 2 quantitative variables when applying allergens to obtain sensitization: the absolute amount of allergen and the surface concentration. This review analyzes the limited studies on surface concentration and determines surface area threshold levels for the allergens documented in the literature.     

Modified short-term guinea pig sensitization tests for detecting contact allergens as an alternative to the conventional test

The conventional adjuvant and patch test (APT) method of guinea pig sensitization testing was modified in 2 ways, s-APT and s-APT(2), in order to shorten the test period. These short-term test methods consist of 72-h closed application of test material with intradermal injection of emulsified Freund's complete adjuvant (E-FCA) for 1st induction, 48-h closed application of test material with (s-APT) or without (s-APT(2)) intradermal injection of E-FCA on the 7th day for 2nd induction, and open application on the 14th day for challenge. They were compared with conventional APT by using 8 allergenic chemicals (formaldehyde, nickel sulfate, cobalt sulfate, ethyl-p-aminobenzoate (benzocaine), isoeugenol, 2-mercaptobenzothiazole, 2,4-dinitrochlorobenzene (DNCB) and 1-phenylazo-2-naphthol (Sudan I)). The short-term methods gave similar results to those of conventional APT in terms of mean response, sensitization rate and sensitization potency (challenge concentration that induces a mean response equal to 1.0). Thus, our short-term methods, which are capable of evaluating skin sensitization within 17 days, are sufficiently sensitive to detect potentially hazardous contact allergens.     

Studies of new short-period method for delayed contact hypersensitivity assay in the guinea pig

A new method for delayed contact hypersensitivity assay of chemical compounds in guinea pigs, a short-period method (14 days) with a high detection sensitivity, has been developed. The new method was as follows; a combination of a Freund's complete adjuvant (FCA, undiluted) intradermal injection and a 24–h occusive patch on a guinea pig was performed 2x at an interval of 4 days and challenged by non-occlusive topical application II days after the first sensitization (with benzyl alcohol during test development). Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dermis were observed histopathologically at the skin reaction site. This newly developed method (adjuvant and 24–h occusive patch 2 test: AP2 test) could equally and/or better detect the allergenicities of 6 other chemical compounds (bromostyrol, citronellal, benzyl salicyfate. p -aminobenzoic acid ethyl ester, phenylenediamine and formaldehyde) as compared with the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT).     

Sensitizing potential of acetaldehyde and formaldehyde using a modified cumulative contact enhancement test (CCET)

The contact allergenic activity of acetaldehyde was investigated with a modified cumulative contact enhancement test (CCET) method in guinea pigs. Possible cross-reactivity between acetaldehyde and formaldehyde was also studied. In contrast to the original CCET protocol, we used sham-treated controls and the chemicals were tested with closed epicutaneous application at 1st challenge. The suitability of the method was verified with formaldehyde and the results were comparable with those previously found with the guinea pig maximization test (GPMT). For the 1st time, acetaldehyde was shown to be a contact allergen in predictive tests. No cross-reactivity was observed between acetaldehyde and formaldehyde. Acetaldehyde seems to be a rare sensitizer in man. However, its allergenic activity should be considered, since it might be present as an impurity in ethoxylated surfactants. As the CCET protocol involves topical induction and challenge, we regard the modified version as well suited to evaluation of the contact allergenic potential of chemicals.     

Studies of new short-period method for delayed contact hypersensitivity assay in the guinea pig

The enhancement effect of cyclophosphamide on the delayed contact hypersensitivity reaction of chemical compounds was studied in Hartley albino guinea pigs. A series of assay procedures. combining the AP2 test (adjuvant and 24-h occlusive patch 2× test, as previously reported) with intraperitoneal cyclophosphamide administration, were examined. The newly developed method was as follows; cyclophosphamide 200 mg/kg intraperitoneal administration 3 days before the 1st sensitization of the AP2 test (cyclophosphamide. adjuvant and 24-h occlusive patch 2× test: CAP2 test). Comparing the CAP2 test with the AP2 test, the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT), the CAP2 test equally and/or better enabled the detection of allergenicities not only of strong allergens such as bromostyrol, citronellal, p -phenylendediamine and formaldehyde, but also of weak allergens such as benzyl salicylate and p -aminobenzoic acid ethyl ester. Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dennis at the skin reaction site were histopathologically observed. Cyclophosphamide effectively enhanced the delayed contact hypersensitivity reaction of weak allergens.     

Oral and epicutaneous desensitization in urushiol contact dermatitis in guinea pigs sensitized by 2 methods of different sensitizing potency

This investigation was conducted to study the relationship between the different intensities of sensitization to urushiol induced by the maximization test method (MT) or by the ear-flank method (EF) in guinea pigs, and hyposensitization induced by repeated oral and/or epicutaneous administration of the allergen, and to compare the immune tolerance achieved between these routes of dosing. During the observation period of 10 weeks, both oral and epicutaneous administration produced hyposensitization to urushiol after weak sensitization by EF; however, only combined administrations via the oral and epicutaneous routes achieved hyposensitization after potent sensitization, while inadequate tolerance was induced by oral dosing and none by epicutaneous dosing. Thus, it is indicated that difficulty in establishing immune tolerance may increase with increasing intensity of primary sensitization, and that hyposensitizing potency tends to increase in the order epicutaneous, oral and combined (e.p.+p.o.) administrations of urushiol.     

Comparison of guinea pig sensitization methods

Up to now, different methods in guinea pig sensitization were used, to assess contact sensitization potential of low molecular weight chemicals. In order to compare the efficiency of the epicutaneous, intradermal and combined sensitization methods, two stronger sensitizers, the water soluble chlormethylimidazoline hydrochloride (CMI) and the lipid soluble 1,3-dinitrochlorobenzene (DNCB) were used for the experiments. Eleven modified sensitization methods were performed. In a first trial each substance was tested on 10 animals. In a further study those techniques which had produced the best results, that is the highest degree of sensitization, were applied to 25 to 30 animals. For comparison of the results of the various methods, a mean sensitization index was calculated. The best results were achieved with the Maximization Test of Magnusson & Kligman (1969) and the Split Adjuvant Technique of Maguire & Chase (1967).     

In vitro study of the cross-sensitivity of hair dye using hapten-specific lymphocytes

Paraphenylenediamine (pPDA), which is used frequently in hair dyes, has a high sensitization potential and is also well-known to cross-react with chemicals of similar structure. pPDA-specific lymphocytes from sensitized inbred guinea pigs were prepared and the cross-reaction of pPDA was examined using cultured epidermal cells as the target. The cytolytic effect was observed to be highest with pPDA, followed by paraaminophenol, orthophenylenediamine, and metaphenylenediamine. Resorcinol and the control produced no cytolytic reaction. These results showed a good correlation between in vitro and in vivo tests. The recognizing ability of hapten-specific lymphocytes is suggested to vary according to the position of the functional group and its type.     

Expression of integrin receptors and ICAM-1 on keratinocytes in vivo and in an in vitro reconstructed epidermis: Effect of sodium dodecyl sulphate

Several integrin heterodimers such as 21, 64 and _v5 are expressed on basal keratinocytes of the epidermis. Because overexpression of these integrins as well as induction of the intercellular adhesion molecule-1 (ICAM-1) have been found in inflammatory dermatoses, we sought to determine whether these modulations could be used as markers of skin irritation. In normal epidermis, topical application of 1% sodium dodecyl sulphate (SDS) for 24 h caused an upregulation of 3, 1, 6, 4, _v, 5 and to a lesser extent 2 integrin chains as well as an induction of ICAM-1. To investigate whether these parameters could also be used for evaluation of skin irritancy in vitro, SDS was applied for 24 h to reconstructed epidermis on de-epidermized dermis (RE-DED). In RE-DED, integrin overexpression and aberrant 5 expression was seen under normal in vitro culture conditions and topical application of SDS caused only marginal additional upregulation. We could not detect any ICAM-1 reactivity on either normal or irritated RE-DED. Our results demonstrate that the modulation of integrin and ICAM-1 expression can be used as markers of irritation of the epidermis in vivo, but not in vitro.This work was supported in part by the Bundesministerium für Forschung und Technik, Bonn, Germany (BMFT: 421-4001-01HK9195).Part of this work was presented at The Second Tricontinental Meeting of the JSID, SID and ESDR, Kyoto, Japan, 28–31 October 1993     

Butenylbithiophene, α-terthienyl and hydroxytremetone as contact allergens in cultivars of marigold (Tagetes sp.)

Ornamental cultivars of Tagetes sp., commonly named marigold, are one of the presently most popular pot, and garden plants. Sensitizing experiments in guinea pigs with short ether extract and isolated compound- repealed the presence of 3 constituents that must be considered as contact allergens. They were identified as 5-(3-buten-1-ynyl)-2.2'-bithiophene, α-terthienyl and hydroxytremetone. In sensitized animals, butenylbithiophene showed moderate to strong Sensitizing potency while α terthienyl was less strong and hydroxytremetone weak. The results demonstrate, for the first time, that at least some of the thiophenes abundantly occurring in many species of the Composite family possess not only phototoxic activity but also sensitizing properties.     

Phosgene (chlorophenyl)hydrazones, strong sensitizers found in yellow sweaters bleached with sodium hypochlorite, defined as causative allergens for contact dermatitis by an experimental screening method in animals

12 young men developed allergic contact dermatitis from wearing yellow cotton sweaters. We attempted to identify the causative agents by an experimental screening method in animals. Guinea pigs were sensitized with an acetone extract of the sweater material, by means of the guinea pig maximization test (GPMT). Active ingredients were then separated from the extract, by step-by-step patch test screening of chromatographic fractions in the guinea pigs, and finally analyzed by gas chromatography-mass spectrometry (GC-MS). Although there were 2 allergens with important activity (1 in the fraction eluted from the silica gel column with hexane, and 1 in the methanol fraction), the present study is focussed on the fat-soluble allergens in the hexane fraction. GC-MS analysis revealed that 4 kinds of phosgene (chlorophenyl)hydrazones (PCPHs) were present in the hexane fraction. PCPHs prepared in our laboratory showed strong eliciting activities, not only in the guinea pigs sensitized with the extract, but also in a male volunteer sensitized by exposure to a yellow sweater during irritancy testing. Phosgene (2,5-dichlorophenyl)hydrazone, which was the main component among the PCPHs found in the sweater, sensitized guinea pigs even at the 1 ppm level. From these results, we conclude that PCPHs were one of the allergens responsible for the cases.     

Identification of contact allergens in C.I. Solvent Red 23 (commercial Sudan III) by chemical analysis and animal testing

C.I. Solvent Red 23, commercial Sudan III, is widely used in cosmetic products. Chemical analyses and guinea pig sensitization tests were carried out to identify its contact allergens. In the Magnusson & Kligman guinea pig maximization test, C.I. Solvent Red 23 showed 20% positive reactions. By conducting chemical analyses with HPLC and GLC, 2-naphthol (82 ppm), azobenzene (48 ppm), Sudan I (570 ppm) and many unknown impurities, as well as the main constituent pigment Sudan III (87%), were found. The chemical structure of one unknown impurity was identified as an isomer of Sudan III. We found that purified Sudan III showed no positive reaction, while the isomer elicited 30% positive reactions, in the same guinea pig test. Furthermore, cross-sensitization with p-phenylenediamine was investigated using the guinea pig test. Animals sensitized with p-phenylenediamine also showed positive elicitation reactions with purified Sudan III. From these results, the contact allergenicity of C.I. Solvent Red 23 is considered to be due to impurities, including the isomer of Sudan III, 1-(o-phenylazophenylazo)-2-naphthol. Positive reactions to Sudan III previously demonstrated in hairdressers are due to cross-sensitivity with p-phenylenediamine.