A method for identifying causative chemicals of allergic contact dermatitis using a combination of chemical analysis and patch testing in patients and animal groups: application to a case of rubber boot dermatitis

A 63-year-old woman developed allergic contact dermatitis from rubber boots. Initial investigation, by patch testing in the patient and chemical analysis of the causative rubber boots, revealed that mercaptobenzothiazole (MBT) and dibenzothiazyl disulfide (MBTS) were the causative chemicals. Subsequent investigations were performed by patch testing in animal groups. An extract of the causative rubber boots, MBT and MBTS were used for sensitization of guinea pigs by the guinea pig maximization test (GPMT). 3 animal groups, A (with the boot extract), B (with MBT) and C (with MBTS) were successfully prepared. The boot extract was fractionated by column chromatography and thin-layer chromatography (TLC). Each fraction was subjected to patch testing in the animal groups. Positive reactions in all groups would show that the active fractions contained MBT-type compounds, whereas a positive reaction in group A but negative ones in group B and C would show that the active fractions did not contain any MBT-type compounds. Each fraction was then analyzed by gas chromatography (GC), GC-mass spectrometry (GC-MS), direct inlet-MS (DI-MS) and high-performance liquid chromatography (HPLC). By this investigation, we found not only known allergens (MBT, MBTS), but also unknown allergens: S-substituted MBT-type compounds and styrenated phenol (SP). Thus, SP was shown to be a candidate as a human sensitizer even though the patient did not react to it.     

Route of contact sensitization and in vitro lymphocyte transformation

A guinea pig skin extract conjugate with dinitrofluorobenzene elicited significant in vitro transformation of cultured lymphnode lymphocytes from 19 of 27 guinea pigs sensitized by footpad injection of dinitrochlorobenzene in Freund's complete adjuvant, as compared to only 1 of 26 guinea pigs topically sensitized to dinitrochlorobenzene. Topically sensitized guinea pigs appear to be more appropriate models for contact allergy in man than guinea pigs sensitized by other methods. Other sensitization procedures are likely to produce more heterogeneous forms of sensitization, with features of contact allergy, tuberculin-type allergy, antibody-mediated hypersensitivity and cutaneous basophile hypersensitivity.     

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.     

Antigen-specific IgG1-mediated epidermal cell injury: a component of contact hypersensitivity reactions in guinea pigs, measurable in vitro in full-thickness skin explants.

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.     

Allergic contact dermatitis to Ginkgo biloba L.: relationship with urushiol

Summary A Ginkgo biloba L. fruit extract was prepared and purified. Three groups of guinea pigs were sensitized to the crude extract, anacardic acids 1, and cardanols 2 respectively, using the FCAT method, and the fourth group to urushiol using the epicutaneous route. Each group was tested for reaction to the primary sensitizer and to the different main aromatic compounds isolated from Ginkgo fruits. Anacardic acids were found to be good sensitizers, while cardanols failed to induce allergic contact dermatitis (ACD). No cross-reactions were observed among the compounds tested. Ginkgolic acids 1 seem to be the main allergens of Ginkgo biloba L. and the hypothesis of a biotransformation of 1 into catechol 4 is not supported by experiment.     

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcal antigens

Summary Staphylococcal homogenate was fractionated into cell walls (CW), cell membranes (CM) (insoluble part left after removal of the cell wall fraction) and a soluble fraction. The capacity of these fractions to evoke delayed skin reactions and to stimulate lymph node and peripheral blood lymphocytes from guinea pigs sensitized with staphylococcal homogenate in Freund's complete adjuvant was tested 21 days after sensitization. Highest skin reactivity was observed with the cell wall fraction. In the lymphocyte stimulation test similar results were obtained with all three fractions. With peripheral blood lymphocytes higher stimulation indices were observed than with lymph node lymphocytes.These investigations were performed under the supervision of the retired director Prof. Dr. H. Storck     

Cross-sensitivity of common aminoglycoside antibiotics.

Guinea pigs were sensitized to neomycin (A, B, or C), paromomycin, gentamicin, kanamycin, streptomycin, and dihydrostreptomycin via intradermal or foot-pad injection with an adjuvant containing killed Mycobacterium butyricum or M tuberculosis H37Ra (Ra). These antibiotics produced greater cross-sensitization with an increase in the number of immunizations and chemical structural similarities. After repeated intradermal injections (adjuvant Ra) of neomycin, guinea pigs showed cross-sensitization to paromomycin, kanamycin, and streptomycin. A single intradermal injection of one of these antibiotics produced stronger reactions to the most closely related antibiotics, with no meaningful sensitization to the least-related allergens. Streptomycin-sensitized guinea pigs seldom showed a meaningful cross-sensitization to dihydrostreptomycin or the other antibiotics (except neomycin C); however, guinea pigs sensitized to dihydrostreptomycin or the other antibiotics often showed strong cross-sensitization to streptomycin.     

An investigation of the irritant and allergenic properties of daffodils (Narcissus pseudonarcissus L., Amaryllidaceae)

Irritancy of daffodil flowers and bulbs was assessed using various fresh plant preparations, solvent extracts and some of the known Amaryllidaceae alkaloids on guinea pigs. Sensitization was also carried out on guinea pigs using these plant preparations, solvent extracts and 7 fractions obtained after preparative chromatography of the bulb ether extract. Only 1 fraction, containing 2 alkaloids, was capable of inducing delayed hypersensitivity in the animals; the sensitivity achieved, however, was weak. The substances were identified as masonin and homolycorin, which acted as elicitors, but masonin may also be a sensitizer. While homolycorin is a known daffodil constituent, masonin has not been found previously in Narcissus pseudonarcissus. 3 other alkaloids as well as chelidonic acid and isorhamnetin were non-elicitors in the sensitized guinea pigs.     

Degradation products of monoterpenes are the sensitizing agents in tea tree oil.

BACKGROUND: Patients using tea tree oil (TTO) topically may become sensitized to this natural remedy. More than 30 cases have been documented in the literature since 1991. OBJECTIVE: Freshly distilled, as well as oxidized TTO, some fractions, and single constituents were used for experimental sensitization in guinea pigs. TTO was stored on a window sill to study the influence of light, oxygen, and warmth. The oxidized oil and different fractions were devoted to experimental sensitization in guinea pigs to determine their sensitizing potency. Fifteen constituents were patch tested in TTO-sensitive patients to find how many may play a role as contact allergens. METHODS: Guinea pigs were sensitized by a modified FCA-method (Freund's complete adjuvant) with freshly distilled TTO, oxidized TTO, the monoterpene and sesquiterpene fraction, and 1, 8-cineole. TTO-sensitive patients were tested with 15 typical constituents and degradation products. Gas chromatographic analysis was used to detect degradation products of the deteriorated TTO. RESULTS: Fresh TTO was revealed to be a very weak sensitizing material whereas oxidized TTO was 3 times stronger. The monoterpene fraction showed to be a stronger sensitizer than the sesquiterpene fraction. All 11 patients reacted mostly with a ++-plus or even a -plus reaction to alpha-terpinene, terpinolene and ascaridol. alpha-Phellandrene became positive in four patients, myrcene in only two. Gas chromatographic analyses showed that the formation of peroxides increased within 4 days from less than 50 to more than 500 ppm. Peroxides, epoxides and endoperoxides were formed. Deterioration products of alpha-terpinene were found to be mainly p-cymene, ascaridol, isoascaridol, a ketoperoxide, and colorless crystals that likely were 1,2,4-trihydroxy menthane. The p-cymene content increased dramatically from 2% to 11.5%. alpha- and gamma-terpinene, as well as terpinolene, were reduced to one half of their former concentration. Ascaridol and isoascaridol have never before been found in TTO. CONCLUSION: Tea tree oil kept in open and closed bottles or other containers undergoes photooxidation within a few days to several months, leading to the creation of degradation products that are moderate to strong sensitizers. Peroxides, epoxides and endoperoxides, like ascaridol and 1,2,4-trihydroxy menthane, are formed. These must be considered responsible for the development of allergic contact dermatitis seen in individuals treating themselves with the oil. A test series with 15 characteristic constituents is recommended for patch testing.     

Study of dose-response relationship in contact sensitivity using an in vitro assay

Dose-response relationships in contact sensitivity were evaluated in guinea pigs using an in vitro assay. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with DNCB and 2,4-dinitrobenzene sulfonic salt (DNBS). Lymph node cells from sensitized and control guinea pigs were cultured in the presence of different doses of DNCB and DNBS at 8 x 10(5) cells/well, respectively. The sensitivity was evaluated by the lymphocyte transformation test (LTT), which was assessed by uptake of 3H-thymidine. The results indicated that there were significant correlations between the doses of sensitizers and the values of LTT in both phases of induction and challenge. Thus, the presence of higher numbers of LTT-reactive lymphocytes in the circulation may well correlate with the sensitizing doses. The values examined by in vitro assay correlated well with patch test readings (r = 0.653), indicating that following the increment of degree of patch test reactions, the values of SI were also increased. The in vitro LTT may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. The in vitro assay reproduced the cross-reaction between DNCB and DNBS which was confirmed in vivo.     

Contact sensitivity to quinidine sulfate from occupational exposure

Three workers exposed to quinidine sulfate became sensitized after short exposure times (2-3 months). They were patch test positive to quinidine in different vehicles, but negative to the diastereoisomer quinine. Guinea pig maximization tests demonstrated quinidine and quinine to be grade V allergens according to the classification of Magnusson & Kligman. When challenged for cross reactivity, the animals sensitive to quinine did not react to quinidine. Among the quinidine-sensitive guinea pigs, three of 20 (P greater than 0.05) reacted when challenged with quinine.     

An attempt to isolate and identify allergens in lanolin

One sensitizer, a sterol, has previously been reported to be present in lanolin. In this study, a related substance, a sterol, with a molecular weight of 424 has been isolated. A few sensitive subjects did not react to this substance. Lanolin probably contains several sensitizers. Sensitization to lanolin in guinea pigs seems previously to have failed. In this study, a methanol extract of a lanolin preparation containing large amounts of sterols sensitized guinea pigs.     

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcus aureus antigens

Summary The development of delayed hypersensitivity against staphylococcal antigens in guinea pigs was observed from day 3 to day 50 after sensitization with staphylococcal homogenate in Freund's incomplete (FIA) and Freund's complete adjuvant (FCA). Skin test reactivity, stimulation of lymph node lymphocytes, and peripheral blood lymphocytes and the titre of precipitating antibodies were followed during this period. Maximal skin test reactivity as well as maximal lymphocyte responsiveness occurred at day 21 after sensitization in FCA-sensitized guinea pigs. In FIA-sensitized animals highest skin reactivity was observed at day 14 and maximal lymphocyte stimulation 35 days after sensitization. Precipitating antibodies reached a plateau at day 20 in plasma of animals sensitized with FCA and at day 35 in FIA-sensitized animals.These investigations were performed under the supervision of the retired director Prof. Dr. H. Storck     

Sensitization of mice to paraphenylenediamine and structurally-related compounds: adjuvant effects of vitamin A supplementation

Allergic contact dermatitis from moderate and weak contact sensitizers is generally studied with guinea pigs, since they are readily sensitized to contact allergens. Mice, by contrast, are poor responders to weak contact allergens. However, the variety of in vitro murine systems us well, is murine specific reagents make mice the preferable species, with the use of vitamin A supplementation, 2 protocols were developed which sensitized CBA/J female mice to paraphenylenediamine, Mice were sensitized by 5 daily topical applications to shasen dorsal skin. Alternately mice were sensitized by 2 intraperitoneal infections of antigen pulsed spleen cells. Sensitization to paraphenylenediamine was determined by ear swelling following topical application. Vitamin A supplementation was found to be essential for optimum response. Lymph node and spleen cell from sensitized mice were capable of proliferating to paraphenylenediamine in vitro. With the use of Vitamin A supplementation and intraperitoneal injection, CBA/J mice were also sensitized to a number of compounds structurally related to paraphenylenediamine, including the ortho- and meta-derivatives of paraphenylenediamine, as well as hydroquinone and resorcinol. These new protocols, combined with vitamin A supplementation, expand the use of mice to study moderate sensitizers with minimal animal utilization.     

Prevention of nickel-induced allergic contact reactions with pentoxifylline

In this study, we investigated the effect of pentoxifylline, an inhibitor of TNF-α, on the contact sensitivity response induced by nickel. For induction, open epicutaneous sensitization by NiSO₄. 6H₂O (25% aq.) solution was applied on the backs of 38 albino guinea pigs 5 days a week for 4 weeks. NaCl (0.9%) solution was applied epicutaneously to 10 albino guinea pigs as a control group. 19 were sensitized by nickel and developed positive patch test reactions. Patch tests were repeated after 10 of the sensitized pigs were given pentoxifylline 20 mg/kg/day orally. At the end of this study, only 2 positive patch test reactions were observed in the pentoxifylline-treated group, while 7/9 of the untreated guinea pigs developed positive reactions. These results suggest that pentoxifylline inhibits the contact sensitivity response induced by nickel only during drug administration.     

Lymphocyte transformation to membrane-conjugated,liposome-conjugated,or unconjugated pentadecylcatechol in the guinea pig

Summary This study examined the in vitro immunogenicity of haptenated liposomes and compared them with haptenated biological membranes and unconjugated hapten. Peripheral-blood lymphocytes were obtained from guinea pigs topically sensitized with pentadecylcatechol (PDC) and immunologically naive guinea pigs. Lymphocyte transformations were studied by [³H]thymidine uptake. PDC failed to stimulate the lymphocytes from the immunologically naive group. There was significant blastogenesis in the cells of the sensitized group, but the degree of stimulation was dependent upon the manner in which the antigen was presented to them. Unconjugated hapten caused a low-level, dose-dependent mitogenesis in the sensitized T cells and hapten-conjugated liposomes enhanced this response (P<0.05). By far the most effective immunogen was a haptenated biologic membrane. In all cases, the mitogenic response was macrophage dependent. It is possible that the haptenated biologic membranes were more effective than synthetic membranes (liposomes) because of the presence of membrane proteins that can conjugate with hapten and from a more effective immunogen.     

Patch testing with beryllium alloy samples in guinea pigs

An experimental study was conducted in guinea pigs for the predictive assessment of the beryllium alloy hazard in occupational exposure of the skin to beryllium compounds. Guinea pigs were sensitized to beryllium sulfate according to the maximized Magnusson and Kligman test, and challenged with beryllium alloys and metallic copper, beryllium and aluminum samples. Results showed a delayed skin hypersensitivity reaction in 30 to 60% of pre-sensitized guinea pigs challenged with copper-beryllium alloys and aluminum-beryllium alloy. An inflammatory follicular reaction was induced by copper in both controls and pre-sensitized guinea pigs.     

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.     

Rôle of a metabolite in allergenicity of Sudan I

A metabolite of Sudan I (l-phenylazo-2—naphthol) was isolated alter in vitro incubation with the supernatant of guinea pig liver homogenate (S·9). The metabolite was found to be 4′-hydroxy-l-phenylazo-2—naplithol by gas chromatography-mass spectrormetry (GC-MS), and proton and carbon-13 nuclear magnetic resonance (NMR) analyses. It elicited positive reactions in guinea pigs sensitized ot Sudan I. It was also shown to be allergenic. The results suggest that para-hydroxylation of the phenyl group of Sudan I may play an important rôle in its allergenicity.     

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.