Experimental nickel sensitization in the guinea pig: comparison of different protocols

3 different sensitization protocols were compared for inducing delayed-type nickel contact hypersensitivity in guinea pigs. Open epicutaneous sensitization (OE) induced nickel allergy in 11/22 (50%) guinea pigs. When intradermal injections of Freund's complete adjuvant into the nickel-painted skin was added to the same protocol. 4/13 (31 %) became sensitized. The guinea pig maximization protocol induced nickel allergy in only 7/31 (23%) of the animals. Compared with the 2 other methods, animals sensitized with open epicutaneous applications reacted more rapidly (maximum at 6 h) and strongly (2+ reaction in 12/22 of animals) in previous patch lest sites upon systemic (i p.) nickel challenge. Open epicutaneous sensitization of guinea pigs should he a useful model for studying cellular and immunological mechanisms in nickel contact sensitivity.     

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.     

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.     

Cross-reactivity to palladium and nickel studied in the guinea pig.

In recent years there have been several reports on concomitant patch test reactions to palladium and nickel, which belong to the same group in the periodic table. Exposure to palladium mainly takes place via dental alloys and jewelry. However, the clinical relevance of simultaneous reactivity to these metals is unknown. To elucidate the question of cross-reactivity, guinea pigs were induced with palladium or nickel and simultaneously challenged with palladium and nickel. Animals sensitized to palladium according to the guinea pig maximization test method (GPMT) or to a new method by van Hoogstraten & Scheper (H&S) reacted to palladium as well as to nickel. On the other hand, animals sensitized to nickel according to H&S reacted to nickel but not to palladium. The GPMT shows that palladium is a more potent sensitizer than nickel: could palladium be the primary sensitizer in humans?     

Frequency of sensitization to methyl aminolaevulinate after photodynamic therapy

Background:  Allergic contact dermatitis to methyl aminolaevulinate (Metvix™) after topical application in photodynamic therapy (PDT) has previously been described in case reports.
Objective:  To compare the frequency of sensitization to Metvix^® cream in a group of patients previously treated at least five times with Metvix-PDT with the frequency observed in an unexposed control group.
Methods:  Twenty patients treated five times or more with Metvix-PDT and 60 controls with no prior exposure to Metvix^® were patch tested with Metvix^® cream and Metvix^® placebo cream. Subsequently, the patients were interviewed to determine the relevance of a positive patch test reaction to Metvix^®.
Results:  Of 20 patients treated with Metvix-PDT, 7 were sensitized to Metvix^® cream, giving a sensation risk of 35%. In the control group, 1 of 60 became sensitized after a single exposure to Metvix^® cream (1.7%). There was no reaction to the placebo cream. The positive patch tests to Metvix^® were considered relevant in four of seven patients (57%).
Conclusions:  This study demonstrates a considerable risk of sensitization after Metvix-PDT. We suggest that the patients are interviewed to detect late or persistent local reactions after PDT. These reactions are often considered to be local infections but may represent allergic contact dermatitis, and therefore, patients should be offered patch testing with Metvix^® cream.     

Sodium tetrachloropalladate (Na2[PdCl4]) as an improved test salt for palladium allergy patch testing

Background:  In the last decades, palladium is widely used in dentistry. Allergic reactions to palladium are rarely diagnosed with patch testing, even when positive results would be expected. Palladium tends to cross-react with nickel, which should give rise to more positive reactions to palladium dichloride (standard test salt).
Objective:  The aim of the study was to test whether or not mono-nuclear sodium tetrachloropalladate (Na₂[PdCl₄]) in petrolatum is a better test salt for diagnosing palladium allergy. Positive reactions to the investigated test salt are compared not only with PdCl_2(aq.), but also to NiSO_4(aq.) and NiSO_4(pet.).
Patients/Methods:  Concentration series of Na₂[PdCl₄] were carried out. 164 consecutive patients were patch tested.
Results:  3% of Na₂[PdCl₄]_(pet.) was found to be the highest non-irritative concentration. The results show ( n  = 164) that Na₂[PdCl₄] covers all reactions to PdCl₂ (1.8%) and provokes more positive reactions (14%). From the 164 patients, 18.3% reacted positively to at least 1 of the nickel salts.
Conclusion:  The sensitivity of patch testing with Na₂[PdCl₄] is increased compared with the PdCl₂ salt. Therefore, it can be concluded that Na₂[PdCl₄] is to be a better test salt for diagnosing palladium allergy with patch testing.     

Reactivity to patch tests with nickel sulfate and fragrance mix in infants

The pattern of patch test reactivity to nickel sulfate and fragrance mix was studied with respect to patch test performance, reproducibility and clinical relevance in a population of unselected infants followed prospectively from birth to 18 months of age. TRUE Test™ patches with nickel sulfate in 3 concentrations, 200, 66 and 22 µg/cm², and fragrance mix 430 µg/cm² were used. A likely case of nickel sensitivity was defined as a reproducible positive reaction with at least homogeneous erythema and palpable infiltration occurring at least 2× and present at both the 12 and 18 months follow-up. 543 infants (268 girls and 275 boys) were tested at least 1×, 304 were tested at both 12 and 18 months. The prevalence of a reproducible positive reaction to nickel was 8.6% (20 girls and 6 boys). A transient positive reaction was observed in 111 children. Clinical relevance of nickel sensitivity was found in only 1 child. No reproducible positive reaction to fragrance mix was found. The high proportion of transient patch test reactivity to nickel sulfate 200 µg/cm² indicates that this standard concentration used for adults cannot be applied to infants. The interpretation of a single positive nickel patch test in infants must be assessed with caution and it is probably of non-specific or irritant nature.     

In vitro and in vivo evaluation of the effect of barrier gels in nickel contact allergy

The protective effect of various ethylenediaminetetraacetate (EDTA) barrier gels on nickel skin penetration was investigated in an in vitro model using human skin. Application of the gels seemed to cause an increased release of nickel from nickel alloys. This nickel did not penetrate the skin barrier but was found to be immobilized on the skin surface. This emphasized the importance of washing the skin surface to remove any surplus of barrier formulation after use, since considerable amounts of nickel will be bound in this formulation. It was found that application of the barrier gels beneath the nickel alloy in contact with the skin significantly reduced the amount of nickel found in the epidermal skin layer. In vivo patch testing with a disc of nickel alloy, with and without use of barrier gel, was performed in 21 nickel-sensitive patients. Patch testing with the nickel alloy without use of barrier gel resulted in positive patch test reactions in 11/21 (52.4%) of the patients tested. Application of a Carbopol gel with 10% CaNa₂-EDTA beneath the nickel disc completely abrogated the allergic contact response in all 21/21 (100%) patients. A Carbopol gel without CaNa₂-EDTA was less effective, inhibiting the response in 15/21 (71.4%). A high concordance was found between epidermal nickel levels found in vitro and the in vivo patch test.     

Pretreatment of the test area with 1-day occlusion improves the response rate to NiSO4 5% pet. Patch tests in subjects with a positive history of nickel allergy

A group of 58 women, aged 18 to 51 years, with a clinical history of nickel allergy, who exhibited equivocal or negative reactions to nickel sulfate 5% pet, patch tests performed on the skin of the back, were recruited consecutively from the patch test clinic from September 1993 to June 19944. In order to improve the response rate to NiSO₄ 5% pet, patch tests, a testing procedure utilizing pretreatment of the test area by 1-day (24-h) occlusion was introduced. Patients underwent 2 patch tests on adjacent sites of the volar surface of both forearms. 3 of the patch tests were performed with 40 mg nickel sulfate 5% pet., whereas a control test was carried out by occluding with an empty chamber. 2 of the nickel sulfate test sites were pretreated with 1-day occlusion performed with an empty chamber. A visual grading system and echographic measurement were used to quantify the responses 30–40 min after patch test removal. Echographic evaluations were carried out using a 20 MHz B-scanner. Measurement of skin thickness and determination of the hypoechogenic dermal area, both considered to be parameters of inflammation, were used to evaluate the intensity of the allergic reaction. At the 3-day (72-h) evaluation. 19 subjects out of 58 clearly showed positive reactions to nickel sulfate 5% pet, at pre-occluded skin sites. Moreover, values of skin thickness and of 0–30 areas at positive pre-occluded nickel test areas were higher in respect to control test areas, confirming clinical evidence of increased response to NiSO₄, after occlusion.     

Pre-treatment of nickel test areas with sodium lauryl sulfate detects nickel sensitivity in subjects reacting negatively to routinely performed patch tests

A fair % of patients with a clinical history of nickel allergy show negative patch test results. To improve the response rate to NiSO₄ 5% pet, patch tests, a testing procedure utilizing pre-treatment of I he lest area by a 24-h application of sodium lauryl sulfate (SLS) was introduced 46 women with a clinical history of nickel sensitivity who exhibited negative reactions to nickel sulfate 5% pet, patch tests. were studied, Patients underwent d patch tests on adjacent sites on the volar surface of the forcarms. 4 patch tests were performed with a 72-h application of 40 mg nickel sulfate 5% pet. While I of these patch tests served as control. 3 test areas underwent 24-h pretreatment with 40 μl SLS. 1 with 0.1% and 2 with 0.5% solution. To evaluate differences in the reactivity to SLS plus nickel sulfate related to the site on the forearm, 0.5% SLS pre-treatment was performed both on a proximal and on a distal lest site. At the 72-h evaluation. 19 subjects out of 46 showed positive reaction to nickel sulfate 5%. At skin sites pre-t railed with SLS. Whereas 23 patients reacted positively at 0.5% SLS pre-treated ureas. Echographic values of skin thickness and of hypo-echogeme dermal areas al positive pre-treated nickel lest. Next higher than al control Jest areas, confirming the clinical evidence of an increased response to NiSO₄ after SLS pre-treatment. The inflammatory reaction, is evaluated clinically and echographically, was much higher al distal skin areas (0.l% SLS and distal (0.5%.) SLS than at proximal 0.5% SLS ones.     

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.     

Cobalt allergy in hard metal workers

Hard metal contains about 10% cobalt. 853 hard metal workers were examined and patch tested with substances from their environment. Initial patch tests with 1% cobalt chloride showed 62 positive reactions. By means of secondary serial dilution tests, allergic reactions to cobalt were reproduced in 9 men and 30 women. Weak reactions could not normally be reproduced. A history of hand eczema was found in 36 of the 39 individuals with reproducible positive test reactions to cobalt, while 21 of 23 with a positive initial patch test but negative serial dilution test had never had any skin problems. Hand etching and hand grinding, mainly female activities and traumatic to the hands, were found to involve the greatest risk of cobalt sensitization. 24 individuals had an isolated cobalt allergy. They had probably been sensitized by hard metal work, while the individuals, all women, who had simultaneous nickel allergy had probably been sensitized to nickel before their employment and then became sensitized to cobalt by hard metal work. A traumatic occupation, which causes irritant contact dermatitis and/or a previous contact allergy or atopy is probably a prerequisite for the development of cobalt allergy.     

Objective assessment of nickel sulfate patch test reactions with laser Doppler perfusion imaging

The laser Doppler perfusion scanning technique was used to assess the superficial blood flow of nickel sulfate hexahydrate patch test reactions. There was good agreement between laser Doppler and visual assessments when the highest assessment values of reactions were studied. Earlier detection of reactions was possible with the laser technique. There was great inter-individual variance in perfusion between identically tested patients. 4 patients were visually negative when a TRUE Test^TM patch test dose of 0.20 mg/cm² was applied for 48 h and the test area read 4× up to 168 h. These 4 showed a dose-related increase in perfusion and visually positive reactions using longer application times. The instrument allowed a dose reduction not possible for visual assessments. Reading transparent patches in contact with the skin through transparent semi-occlusive plastic foil or through windows in the tape strip over the patches, allowed us to detect perfusion at 48 h, where a longer application would have been needed using tape and visual assessments.     

Value of the cutaneous basophil hypersensitivity (CBH) response for distinguishing weak contact sensitization from irritation reactions in the guinea pig

Numerous studies of the histology of allergic contact dermatitis reactions to potent allergens in guinea pigs and humans have indicated that there is significant tissue infiltration with basophilic leukocytes. In this study we determined whether this histologic finding could be of value in distinguishing weak sensitization reactions from primary irritation, thereby aiding in the predictive identification of weak or moderate contact allergens. Guinea pigs were sensitized by the Buehler test method. Skin reactions were graded 24, 48, and 72 h post-challenge with duplicate patch sites biopsied at the 24- or 72-h grading timepoints. The biopsies were fixed, embedded in glycol methacrylate, thin sectioned, and Giemsa stained. The number of basophils per 400 leukocytes were counted along the upper dermis just below the dermal/epidermal junction. Challenge patch sites from animals sensitized to a relatively low dose of the strong contact allergen, oxazolone, were compared with patch sites from animals challenged only with a strong irritant, sodium lauryl sulfate (SLS). Compared to normal skin (7.5 +/- 1.0 basophils/400 leukocytes +/- SEM) only the oxazolone patch sites showed significant basophil infiltration (36.8 +/- 6.5), despite the fact that the skin reactions to the low oxazolone challenge dose were relatively weak. SLS patch sites showed no basophil infiltration above normal skin levels (4.8 +/- 0.9). Subsequent blinded studies compared weak/moderate presumptive sensitization reactions (as defined by accepted visual skin grading criteria) to various chemicals (citronellal, vanillin, cinnamic aldehyde, and ethylenediamine) to primary irritation reactions to the same chemicals. In each case, low-challenge-dose sensitization sites on previously treated (induced) animals showed mean basophil infiltration (range, 11.9-69.2 basophils/400 leukocytes) significantly greater than higher-dose irritant reactions (range, 1.6-13.3). The range for normal skin was 0.2-10.2 and the range for strong patch reactions to higher concentrations of oxazolone was 59.8-209.3. These data strongly indicate that light-microscopic quantitation of the CBH response can be used to distinguish relatively weak to moderate contact sensitization reactions from primary irritation reactions to the same chemicals.     

Patch test results with serial dilutions of nickel sulfate (with and without detergent), palladium chloride, and nickel and palladium metal plates

Clinical experience suggests the existence of different degrees of sensitivity in nickel-allergic patients. For quantification of this phenomenon, 462 consecutive patients with previously diagnosed or strongly suspected nickel allergy were tested with serial dilution patch tests with 5 ppm to 5% nickel sulfate in pet. (Ni), and 5 ppm to 1% nickel sulfate in pet. with 1% detergent (Ni/D). Additionally, nickel and palladium metal plates were tested in 103, and cobalt salts, dichromate and palladium chloride (PdCl₂) in most patients. 332 patients reacted positively to Ni or Ni/D. The influence of a concomitantly administered detergent was not significant. A significant correlation was found between positive reactions to low concentrations of Ni (or Ni/D), i.e., 0.1% or less ( N =166), and concomitant reactions to nickel metal plates, cobalt salts and PdCl₂ and a history of ear piercing with metal intolerance. The clinical relevance of reactions to PdCl₂ is at present not clear. A subgroup of nickel-allergic patients with "high sensitivity" can be defined. In future studies further addressing the clinical relevance of high versus low sensitivity, patch testing with 0.01, 0.1, 1.0 and 5% nickel sulfate in pet is recommended instead of routine tests with 5% only.     

The dose–response relationship between the patch test and ROAT and the potential use for regulatory purposes

Background: Allergic contact dermatitis is common and can be prevented. The relationship between thresholds for patch tests and the repeated open application test (ROAT) is unclear. It would be desirable if patch test and ROAT data from already sensitized individuals could be used in prevention.
Objectives: The aim was to develop an equation that could predict the response to an allergen in a ROAT based on the dose–response curve derived by patch testing.
Materials/methods: Results from two human experimental elicitation studies with non-volatile allergens, nickel and the preservative methyldibromo glutaronitrile (MDBGN), were analysed by logistic dose–response statistics. The relation for volatile compounds was investigated using the results from experiments with the fragrance chemicals hydroxyisohexyl 3-cyclohexene carboxaldehyde and isoeugenol.
Results: For non-volatile compounds, the outcome of a ROAT can be estimated from the patch test by: ED_xx(ROAT) = 0.0296 ED_xx(patch test). For volatile compounds, the equation predicts that the response in the ROAT is more severe than the patch test response, but it overestimates the response.
Conclusions: This equation may be used for non-volatile compounds other than nickel and MDBGN, after further validation. The relationship between the patch test and the ROAT can be used for prevention, to set safe levels of allergen exposure based on patch test data.     

Is the variability of nickel patch test reactivity over time associated with fluctuations in the systemic T‐cell reactivity to nickel?

Background  Patch test reactivity to nickel varies over time. To what extent this variation is associated with fluctuations in the T-cell reactivity to nickel is not known.
Objectives  Our aim was to investigate the relationship between variation over time in the patch test and the systemic T-cell reactivity to nickel.
Methods  Patients ( n  =   15) with a history of contact allergy to nickel were subjected to three consecutive patch tests at 3-month intervals, utilizing NiSO₄ at 10 concentrations ranging from 0·0032% to 12·5%. Prior to each patch test, blood mononuclear cells were analysed for T-cell reactivity to nickel by interleukin (IL)-4 and IL-13 enzyme-linked immunospot assay.
Results  Eleven patients reacted positively in all three patch tests, two patients reacted in one or two tests and two remained negative. All 13 positive patients displayed variability over time, in terms of the lowest dose of nickel to which they responded. Also the cytokine response to nickel varied over time but the patients' mean cytokine response was positively correlated with their mean patch test reactivity ( r _s = 0·70, P  <   0·01 for IL-4; r _s = 0·78, P  <   0·001 for IL-13). However, although the changes over time in patch test reactivity and the cytokine responses to nickel displayed a similar pattern in many patients, there was no significant correlation between the individuals' variation over time in vivo and in vitro .
Conclusions  The overall magnitude of the T-cell reactivity to nickel and the patch test reactivity are closely associated but fluctuations in the systemic T-cell reactivity cannot be singled out as the major cause of longitudinal variability in nickel patch test reactivity.     

Contact sensitivity to acrylate compounds in guinea pigs

As reports of contact dermatitis in humans due to acrylate compounds have increased considerably in recent years, it was decided to investigate the ability of these chemicals to evoke contact sensitivity skin reactions in guinea pigs. 21 different acrylate and methacrylate compounds were scanned for their ability to induce contact sensitivity, using 5 different sensitization protocols. Contact reactions of varying intensities were produced to all the mono-, di- and triacrylates tested. However, it was not possible to sensitize guinea pigs to any methacrylates. It would appear that guinea pigs cannot be contact sensitized to acrylate chemicals that are substituted on carbon 2.     

Open, closed and intradermal testing in nickel allergy

Open, closed and intradermal testing with NiCl₂ was performed in 15 subjects with patch-test-proven allergy to 5% NiSO₄ in pet. Intradermal testing proved to be a reliable method in confirming nickel sensitivity within 24 h. Open testing with non-toxic concentrations of NiCl₂ in alcohol resulted in 73% and 93% positive reactions at 24 h and 48 h readings, respectively. This test method can be used as a reliable screening method in nickel allergy. Open testing often resulted in positive reactions within a few hours. This makes it possible to investigate pathogenetic events of acquired allergic contact dermatitis at a much earlier stage than with the usual 48-h occlusion. 24-h occlusion with Finn Chambers is not sufficient if one is to avoid false negative reactions in nickel allergy. Occlusion with Finn Chambers seems to delay the reaction.     

p-Phenylenediamine sensitization is more prevalent in central and southern European patch test centres than in Scandinavian: results from a multicentre study

Background:  Positive patch test reactions to p -phenylenediamine (PPD) are common. PPD is used in oxidative hair dyes and is also present in dark henna temporary 'tattoos'. Cross-sensitization to other contact allergens may occur. Because subjects sensitized to PPD are at risk of clinically severe reactions upon hair dyeing, there is a need for 'current' prevalence data on PPD sensitization.
Objectives:  To compare PPD patch test results from dermatitis patients tested between 2003 and 2007 in 10 European patch test centres and to analyse the causes and determine relevance of positive PPD patch test reactions.
Materials:  Patch testing was performed using PPD (1% free base in petrolatum from Trolab (Almirall Hermal GmbH, Reinbeck, Germany) or Chemotechnique (Malmö, Sweden), equivalent to 0.090 mg/cm² in the TRUE^® test from MEKOS Laboratories AS). Statistical analysis was performed using the chi-squared test.
Results:  The weighted average prevalence was 4.6% among 21 515 patients. PPD sensitization occurred more often in centres located in Central and Southern Europe than in Scandinavian centres (odds ratio = 2.40; 95% confidence interval = 2.07–2.78). The overall proportion of positive patch test reactions to PPD that were registered as being of either current or 'past' relevance was high (weighted average 53.6% and 20.3%, respectively). Consumer hair dyeing was the most prominent cause of PPD sensitization (weighted average 41.8%). Furthermore, occupational hair dye exposure (10.6%) and cross-sensitization to textile dyes (12.6%) were frequently reported.
Conclusions:  PPD sensitization caused by exposure to hair dyes is frequent and remains a present problem for patients visiting contact dermatitis clinics, especially in patch test centres located in Central and Southern Europe.