Effects of aluminium chloride added to common patch test substances

A modulating effect of aluminium regarding type IV reactions might exist but has not been further investigated.


| INTRODUCTION
Patch testing remains the gold standard when establishing the diagnosis of allergic contact dermatitis. The test is performed in vivo by applying potential allergens under occlusion on the skin under standardized conditions. Development of an eczematous reaction, at the site of exposure, makes the test positive for that specific allergen. 1 Having a standardized patch test procedure enables the clinician to draw correct conclusions when defining whether the found allergy is of clinical relevance. It is thus important to add the right amount of antigen in the different tests upon patch testing. The European Society of Contact Dermatitis has developed a European baseline patch test series, with common and clinically well-defined allergens. 1 The doses of allergens are based on clinical experience, traditions, and systematic investigations, [2][3][4] with the ambition of optimizing the sensitivity and specificity, and of lowering the risk of active sensitization. 1 A too-low concentration of a substance in a hypersensitive person might not activate the immune response, yielding a false-negative result. A high concentration can not only give the allergic patient a strong eczematous reaction, but also increase the risk of sensitizing a patient not previously allergic to the tested substance. Furthermore, some allergens might have an irritating effect on the skin, leaving it red and itchy, although no contact allergic reaction is actually present. For the dermatologist, particularly unexperienced ones, these reactions can be difficult to read, and there is a risk that they are read as positive; hence, they become falsepositive reactions.
Optimizing the sensitivity and specificity of patch testing is an ongoing process. Methods that have been described previously include increasing the penetration of allergen through stratum corneum (tape stripping) or irritating the skin and thereby enhancing the innate immune system, such as creating a skin occlusion 24 h before patch testing, or pre-treating the skin with sodium laureth sulphate. [5][6][7][8][9] Aluminium is known for its ability to activate the immune system when used as an adjuvant in vaccines. 10 As contact allergy might occur as a side effect of vaccines with aluminium as adjuvants, there are studies on sensitized individuals in which delayed hypersensitivity reactions have been studied. 11,12 A modulating effect of aluminium with regard to type IV reactions might thus theoretically exist but has not been further investigated.
The aim of this study was to investigate the effect on patch test reactions when adding aluminium chloride hexahydrate (Al-Cl) to test preparations with common allergens. Therefore Al-Cl was mixed with nickel sulphate hexahydrate (Ni), and thereafter we found it interesting to see the effect when adding Al-Cl to preparations with allergens that can be found in consumer products, such as methylisothiazolinone (MI) and the components in fragrance mix I (FM I).

| Study design and participants
At the Department of Occupational and Environmental Dermatology in Malmö, Sweden, three studies were performed. Initially, a pilot study (study 1) was conducted with consecutive dermatitis patients who were patch tested with the Swedish baseline series, our extended baseline series and five additional patches containing a mix of Ni 15.0 w/v % aqua (aq) with different concentrations of Al-Cl (Table 1).
After this pilot study, two more studies were carried out. One study had participants already known to have contact allergy to MI (study 2), but no allergy to Al-Cl, and the other study had participants already known to have contact allergy to FM I but no allergy to Al-Cl (study 3).
At the department, all patch test data and basic characteristics of patients are filed in our computer system Ekta. For studies 2 and 3, we extracted data from all patients with an allergy to FM I 8.0% petrolatum (pet) or MI 0.2% aq and no allergy to Al-Cl 10.0% pet from 2016 to 2021. From these lists, patients were contacted by telephone and invited to participate in the studies. We paid notice to the patient's original patch test reaction to the allergen, and made sure that participants with +, ++ and +++ were all represented in the studies.
Exclusion criteria were age < 18, not able to understand or follow instructions in Swedish, known pregnancy, immunosuppressive treatment, and no contact allergy to Al-Cl 10% pet.
Controls were also enrolled in the studies. These were patients who had been patch tested at the department during the past 6 months without any positive reactions to Al-Cl, MI or to any perfume allergen. These persons were contacted in the same way as T A B L E 1 Substances and concentrations of the different mixes with Al-Cl used in the three studies.  Control-4 0 20 Control-5 0 30 mentioned above. Seven controls were tested with the MI mixes and 10 with the FM I mixes.

| Ethics
The study was approved by the Regional Ethical Review Board, Lund Sweden, dnr 2020/00358 for the pilot study and dnr 2021/01420 for the studies with MI and FM I.  The patches were removed from the skin after 2 days and read- McNemar's test was conducted in study 1. The test substance without Al-Cl (Ni-aq and Ni-pet) was compared with the mixes containing Al-Cl to investigate which mix had the highest sensitivity.

| Patch test preparations and patch test method
In studies 2 and 3, Fisher's exact test, two-sided was used to compare any differences between allergic participants and controls.
Wilcoxon signed-rank test was used to calculate pairwise comparison in STS for the different mixes.

| Study 1: Ni and Al-Cl
One hundred and twenty consecutive dermatitis patients were included in the pilot study. Thirty-two patients were positive to one or more of the different Ni-preparations, listed in Table 1, without being positive to Al-Cl 10.0% pet in the extended baseline series. One patient was only positive to Ni-Al30 and Ni-Al10 on D7 (patient nr 23, Table 2). All other patients were positive to the Ni-preparations already on D3. For the 16 patients positive to Ni-pet, we calculated STS for all mixes, Figure 1, and found that Ni-Al30 and Ni-Al20 gave significantly higher STS than Ni-aq and Ni-pet (Wilcoxon signed-rank test, p value < 0.05 for both mixes).

| Study 2: MI and Al-Cl
One participant had a ++ reaction and one participant had a + reaction to control patch 5 (Al-Cl 30%) on D3. Both participants did not show stronger reactions to MI-Al30 or MI-Al20 than to MI-aq. However, we decided to exclude both participants from the calculations.
We therefore ended up including 20 participants with known allergy to MI. None of the controls reacted to any of the mixes or the control patches with Al-Cl, Fisher's exact test (two-sided), p value < 0.001.
Among the participants with known contact allergy to MI, 16 and MI-aq. Seventeen out of 20 (85%) reacted to MI-Al10. Figure 2 shows the STS for the MI-mixes. MI-aq had significantly higher STS than MI-Al20 and MI-Al10, but no difference was found between MI-Al30 and/or MI-Al5 and MI-aq (Wilcoxon signed-rank test, p value = 0.015 for both mixes).  Figure 3 (Wilcoxon signed-rank test, p value > 0.05).

| DISCUSSION
In the baseline patch test series, some mixes have been developed to save space on back of the patients. The mixes often contain the allergens in lower concentrations than when the individual allergens are patch tested separately. We know from the patch test results with mixes that the combination of substances may enhance penetration, co-react within the vehicle, and thus increase the reaction or possibly modulate the immune reaction within the skin. 16 The pilot study on consecutive patients was performed to investi- Al-Cl 10% pet have the highest sensitivity. Higher and lower concentrations detect fewer allergies. 12 As no contact allergy to Al-Cl 10% pet was found among the 32 patients, we could not interpret the increased reactivity as a synergistic or additive effect of two allergens. [16][17][18][19] The morphology of the reactions to Al-Cl 10% pet and to the Ni-Al preparation did not indicate irritative reactions, hence an additive effect of an irritative reaction from Al-Cl seems not to be the explanation. 5,6,8,20,21 As aluminium in other forms, such as aluminium potassium sulphate, aluminium hydroxide, and aluminium phosphate, is known for its ability to activate the immune system when used as adjuvant in vaccines, 10   Chemistry studies about metal ions binding to proteins have shown that concomitant exposure to ions of chrome and/or aluminium together with cobalt or nickel ions lowers the elicitation threshold of allergic reactions. 27 Hence, what we see in study 1 might be the result of an increased penetration due to chloride ions together with an altered metal ion binding to proteins, leading to increased aggregations and immune activations compared to proteins that are exposed to Ni alone. 27 This could also explain why we get such different results with MI and FM I, as these substances are not metals.
To develop adjuvants in patch testing could be important, when very weak but still symptomatic contact allergies cannot be detected with our standardized concentrations. Increasing the dose is not always an optimal choice, as risks for irritancy and active sensibilization increase. The field needs understanding of the immunohistochemistry actions of specific allergens as well as the interactions of adjuvants and allergens, and different adjuvants might be needed to different test substances.