Contact Allergenicity Research Articles

Contact Allergy in Western Sweden to Propolis of Four Different Origins.

Propolis, which is produced by honeybees and is used in “natural” products, can cause contact allergy. The composition of propolis varies between regions, but little is known about how this variation affects contact allergenicity. The aims of this study were to investigate the frequency of propolis contact allergy in western Sweden, and whether the frequency varies according to the origin of the propolis. Patch-testing was performed using propolis from China, Lithuania, North America, and Sweden in 722 consecutive patients with dermatitis in western Sweden. Frequencies of positive patch-test reactions ranged from 2.4% to 3.6%. There were some, not statistically significant, differences in frequency of contact allergy to the 4 samples of propolis of different origins, with the highest frequency to the sample from China and the lowest frequency to the sample from Sweden. Concomitant positive patch-test reactions to plant and fragrance substances in the baseline series were common, most frequently to Myroxylon pereirae resin and colophonium.

A plasmacytoid dendritic cell (CD123+/CD11c−) based assay system to predict contact allergenicity of chemicals

A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c−) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens ( N = 26) or non-allergens ( N = 22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥1.5-fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.

The human skin/chick chorioallantoic membrane model accurately predicts the potency of cosmetic allergens

The current standard method for predicting contact allergenicity is the murine local lymph node assay (LLNA). Public objection to the use of animals in testing of cosmetics makes the development of a system that does not use sentient animals highly desirable. The chorioallantoic membrane (CAM) of the chick egg has been extensively used for the growth of normal and transformed mammalian tissues. The CAM is not innervated, and embryos are sacrificed before the development of pain perception. The aim of this study was to determine whether the sensitization phase of contact dermatitis to known cosmetic allergens can be quantified using CAM-engrafted human skin and how these results compare with published EC3 data obtained with the LLNA. We studied six common molecules used in allergen testing and quantified migration of epidermal Langerhans cells (LC) as a measure of their allergic potency. All agents with known allergic potential induced statistically significant migration of LC. The data obtained correlated well with published data for these allergens generated using the LLNA test. The human-skin CAM model therefore has great potential as an inexpensive, non-radioactive, in vivo alternative to the LLNA, which does not require the use of sentient animals. In addition, this system has the advantage of testing the allergic response of human, rather than animal skin.

Contact Allergenicity and Cross Reactivity of 2,4,5,6-Tetrachloroisophthalonitrile in a Modified Guinea Pig Maximization Test

Contact Allergenicity and Cross Reactivity of 2,4,5,6-Tetrachloroisophthalonitrile in a Modified Guinea Pig Maximization Test

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Only MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.

Contact Allergenicity of Methylene-bis(thiocyanate) in the Guinea Pig Maximization Test

Contact Allergenicity of Methylene-bis(thiocyanate) in the Guinea Pig Maximization Test

Selegiline Transdermal System (STS): Assessments of Dermal Safety in Human

The selegiline transdermal system (STS) is being developed to treat major depressive disorder. In a Phase I clinical study, the STS (20 mg/20 cm 2 ) produced mild dermal irritability and demonstrated a low potential for contact allergenicity. In Phase III multicenter studies in major depression, over 1800 patients have been evaluated for dermal safety to reveal an overall rate of application site reactions (ASRs) with STS of 21.8% compared with a placebo rate of 9.7%. Discontinuation rates due to dermal adverse events in clinical studies were low (3.5%). Overall, the STS demonstrated good tolerability regarding ASRs and the potential for delayed contact hypersensitivity.

Allergenicity evaluation of Bioban CS-1135 in experimental animals.

An industrial preservative, Bioban CS-1135, was evaluated for its contact allergenicity by means of multiple-dose guinea-pig maximization test and non-radioactive murine local lymph node assay. In the guinea-pig test, an induction dose of 0.5% Bioban CS-1135 sensitized all animals of the group. The dose-response study of the elicitation phase determined a minimum elicitation dose of 5% for positive skin reactions. In the murine assay, Bioban CS-1135 at doses of 10% and more exerted significant effects on lymphoid cell proliferation. Although the data clearly designated Bioban CS-1135 as a skin sensitizer, its relative potency was ranked lowest among skin-sensitizing biocides previously evaluated in this laboratory.

A Two-centre Evaluation of the Human Organotypic Skin Explant Culture Model for Screening Contact Allergens

Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.

Allergenicity Evaluation of N-(1-Methylheptyl)-N'-Phenyl-p-Phenylenediamine and 2-(Thiocyanomethylthio) Benzothiazole by the Guinea Pig Maximization Test.

In order to regulate contact sensitizers in commercial products, not only is identification of chemical allergens essential but also quantitative evaluation of their allergenicity. We have so far reported threshold values of chemical allergens for both induction and challenge phases in the modified guinea pig maximization test (GPMT). In this study, we examined N-(1-methylheptyl)-N′-phenyl-p-phenylenediamine (MHPPD), an antioxidant for rubber products, and 2-(thiocyanomethylthio) benzothiazole (TCMTB), a biocide for plastic and wood products. MHPPD and TCMTB exhibited distinct profiles of contact allergenicity with the GPMT; that is, both could sensitize guinea pigs by the second induction procedure of topical application alone, even without the first induction procedure of intradermal injection. The calculated challenge threshold values for MHPPD and TCMTB were 0.9 and 0.8 ppm, respectively, which were at least one tenth lower than those of chemical allergens previously examined by us. Octanol-water partition coefficients (log p) used as an index of skin permeability were not necessarily higher for MHPPD and TCMTB than those of other allergens, thus, the skin permeability of these compounds did not seem to be related to their ability to cause sensitization by topical application alone or their low challenge threshold values. The results suggest that the sensitizing potential of both compounds is due to their molecular basis. Cross-reactivities among homologues of both allergens were examined to evaluate their antigenic determinants.

Murine local lymph node assay for predictive testing of allergenicity: two irritants caused significant proliferation.

The murine local lymph node assay is a method for predictive testing of contact allergenicity, but its ability to discriminate between allergens and irritants has been questioned. To explain some of the conflicting results with irritants, the proliferation induced by methyl salicylate and nonanoic acid, both considered to be non-sensitisers, was further investigated. Both substances showed a dose--response relationship and clearly positive results when tested at higher concentrations (> or = 50%) and would thus be classified as potential sensitisers according to the present criteria for a positive assay result. In the case of methyl salicylate, the use of either dimethyl formamide or methyl ethyl ketone as vehicle did not significantly influence the results. The negative results obtained for methyl salicylate in some earlier reports were probably due to testing at too low concentrations. The proliferation induced by irritants such as methyl salicylate and nonanoic acid and inter alia sodium dodecyl sulfate, Triton X-100, oxalic acid, chloroform/methanol (2:1) must be better recognized and elucidated before the assay can be generally accepted as a predictive test method.

A modification of the local lymph node assay for contact allergenicity screening: measurement of interleukin-2 as an alternative to radioisotope-dependent proliferation assay

The local lymph node assay is an effective prediction method for contact allergenicity, but employs radioisotopes. We investigated whether measurement of interleukin-2 (IL-2) production by lymph node cells could be used instead to predict contact allergenicity of chemicals. Test chemicals were applied for three consecutive days to both ears of Balb/c mice and the auricular lymph nodes were obtained on either the fourth or fifth day after the first application. Both IL-2 concentration in supernatant of the suspension and proliferative activity of lymph node cells were determined after 24-, 48-, 72-h cell culture in RPMI-1640 medium by ELISA and by measuring [ 3H]methylthymidine incorporation, respectively. These two methods detected allergenicity similarly except in the case of TNCB and oxazolone, which showed excessive proliferation-inducing capacity as compared to IL-2 release-increasing effect. Flow cytometry showed that these two chemicals also increased the percentage of Ia d-positive cells in the lymph nodes, suggesting that these chemicals might induce not only cellular immunity but also humoral immunity. We conclude that interleukin-2 assay is a convenient and dependable method for screening strong contact allergens without using radioisotopes.

Contact Allergenicity and Cross-Reactivity among 2-Mercaptobenzimidazole, Methyl 2-benzimidazolecarbamate and Ethylene Thiourea for Use in House Products.

Contact Allergenicity and Cross-Reactivity among 2-Mercaptobenzimidazole, Methyl 2-benzimidazolecarbamate and Ethylene Thiourea for Use in House Products.

Induction of type IV hypersensitivity to contact allergens in guinea pigs by in vitro haptenized allogenic peritoneal exudate cells.

The induction of type IV hypersensitivity to contact allergens in guinea pigs has been studied by using allogenic peritoneal exudate cells (> 90% macrophages), which had been incubated primarily in vitro with dinitrochlorobenzene, formaldehyde, potassium dichromate, nickel II sulphate or para-aminobenzoic acid. In these guinea pig sensitization experiments Freund's complete adjuvant was used. In all haptens investigated the sensitization rates of the presented method were parallel to the known contact allergenicity in humans and, apart from the potassium dichromate results, comparable with those of the guinea pig maximization test. Because of its alternative immunization procedure, in which only few or no allergen molecules escape the effective presentation pathway, the authors conclude that this method could be developed into a predictive test assay for the evaluation of the contact allergenicity of water-soluble substances.

Challenge assay in vitro using lymphocyte blastogenesis for the contact hypersensitivity assay

To confirm positivity in routine guinea pig studies, contact allergenicity was investigated by a challenge assay in vitro using a co-culture of autologous lymphocytes passed through a nylon-wool column and antigen-presenting cells (APCs) modified with or without antigen. Proliferation of the lymphocytes primed with ovalbumin and/or 2,4-dinitrochlorobenzene was antigen specific and dependent on the presence of APCs (peripheral blood monocytes, splenic macrophages and macrophages induced by liquid paraffin). For another nine haptens, primed lymphocytes proliferated significantly more than control lymphocytes; the stimulation index (SI; ratio between [ 3H]methylthymidine ([ 3H]TdR) incorporation of lymphocytes with antigen-modified APCs and [ 3H]TdR incorporation of lymphocytes with APCs not modified by antigen) was 1.6–4.8 in sensitized animals whereas it was about 1.0 in control animals. Sodium dodecyl sulfate did not cause lymphocyte proliferation. The SI value in vitro was correlated with both the positive rate in vivo ( r = 0.736) and the mean response score in vivo ( r = 0.645). Thus, it was possible to confirm that positivity in routine experiments was a true sign of allergy. A combination of this assay and short-term animal studies would provide an efficient assessment of the allergic potential of chemicals.

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.

1-Chloromethylpyrene: a reference skin sensitizer and genotoxin

1-Chloromethylpyrene (1-CMP) has been evaluated as a model mutagen and toxin related to the ultimate electrophiles derived from benzo[ a]pyrene and 1-nitropyrene. It was mutagenic to Salmonella (> 100 pg/plate) an exceptionally reactive to DNA when assessed by the 32P-postlabelling technique. 1-CMP was inactive in a mouse bone micronucleus assay when administered by gavage, probably due to hydrolysis, whose kinetics have been studied ( t 1 2 ∼ 23 min at 37°C). However, as expected, it was a potent skin toxin as determined by its activity as a mitogen to mouse skin and its contact allergenicity, as determined using the local lymph node proliferative assay. It is concluded that 1-CMP will probably be a potent human skin carcinogen and contact allergen.

Are free radicals and not quinones the haptenic species derived from urushiols and other contact allergenic mono- and dihydric alkylbenzenes? The significance of NADH, glutathione, and redox cycling in the skin

The induction of allergic contact dermatitis to urushiols from poison ivy and related plants is generally believed to involve an initial oxidation event by which a protein-reactive quinone is formed. However, this does not readily account for the contact allergenicity of closely related mono- and dihydric alkylbenzenes such as the alkylphenols and alkylresorcinols which are not so easily oxidised to quinones in vitro. When the redox processes known to occur in living tissues are taken into consideration, a more plausible unifying mechanism involving the formation of protein-reactive radical species becomes apparent. Experiments described here examine the autoxidation of p-benzoquinone and various mono- and dihydric benzenes and alkylbenzenes, and their reactions with the diphenylpicrylhydrazyl radical, cysteine, glutathione, and NADH. We have also demonstrated that administration to mice of 2-oxo-4-thiazolidine carboxylate, a compound known to elevate intracellular glutathione levels, inhibits the irritancy and sensitising activity of 3-pentadecylphenol. This work suggests that redox cycling in the skin following penetration of allergenic mono- and dihydric alkylbenzenes initially depletes local levels of endogenous reducing equivalents such as glutathione and NADH; once depleted, further cycling results in the uncontrolled generation of radical species which may reasonably be expected to exhibit protein reactivity.

Final Report on the Safety Assessment of Octyl Palmitate, Cetyl Palmitate and Isopropyl Palmitate

The Palmitates used in cosmetic products are esters of palmitic acid and octyl, cetyl, or isopropyl alcohol. The acute oral LD50 is estimated from studies with rats to be greater than 14.4 g/kg for Cetyl Palmitate and greater than 64.0 g/kg for Octyl and Isopropyl Palmitates. Acute studies with rabbits showed no evidence of dermal toxicity for any of the Palmitates. Isopropyl Palmitate was “well tolerated” and Octyl Palmitate was nontoxic in separate subchronic dermal studies. Rabbit skin tests with the Palmitates showed that they are nonirritating and nonsensitizing. Also, Draize rabbit eye irritation tests on the Palmitates produced either no or only very slight ocular irritation. One of three formulations containing Octyl Palmitate at concentrations between 40% and 50% produced mild irritation. Formulations containing Cetyl Palmitate at concentration of 2.7% were minimally irritating and produced no signs of sensitization, phototoxicity, or photo contact allergenicity. A formulation containing 45.6% Isopropyl Palmitate produced no signs of irritation, sensitization, phototoxicity, or photo contact allergenicity. From the available information, it is concluded that Octyl Palmitate, Cetyl Palmitate, and Isopropyl Palmitate are safe as cosmetic ingredients in the present practices of use and concentration.

Mechanism for 6-methylcoumarin photoallergenicity

6-Methylcoumarin (6-MC), a synthetic fragrance material, has been reported to be photoallergenic both in man and in guinea pigs. To elucidate the possible mechanism of photoallergenicity, 6-MC in ethanolic solution was exposed to 200 joules/cm 2 of long-wavelength ultraviolet (UV) ranging from 320 to 400 nm for 16 hr, and was examined for its contact allergenicity by means of a modified guinea pig maximization test. Strong allergic responses were observed, indicating that UV plays a catalytic role in forming contact allergens during irradiation. The solution was fractionated to isolate and purify contact sensitizers by gel-permeation chromatography and high-performance liquid chromatography. By employing mass spectrometry and proton and carbon-13 nuclear magnetic resonance, mono- and diethyl esters of 6-MC dimer were compounds identified which showed allergic reactions in guinea pigs sensitized with UV-irradiated 6-MC.