European patch test results with audit allergens as candidates for inclusion in the European Baseline Series, 2019/20: Joint results of the ESSCAA and the EBSB working groups of the ESCD, and the GEIDACC

In 2019, a number of allergens (haptens), henceforth, “the audit allergens,” were considered as potential additions to the European Baseline Series (EBS), namely, sodium metabisulfite, 2‐bromo‐2‐nitropropane‐1,3‐diol, diazolidinyl urea, imidazolidinyl urea, Compositae mix II (2.5% or 5% pet), linalool hydroperoxides (lin‐OOH), limonene hydroperoxides (lim‐OOH), benzisothiazolinone (BIT), octylisothiazolinone (OIT), decyl glucoside, and lauryl glucoside; Evernia furfuracea (tree moss), was additionally tested by some departments as well.

some departments as well.
Objectives: To collect further data on patch test reactivity and clinical relevance of the audit allergens in consecutive patients across Europe.
Methods: Patch test data covering the audit allergens in 2019 and 2020 were collected by those departments of the European Surveillance System on Contact Allergies testing these, as well as further collaborators from the EBS working group of the European Society of Contact Dermatitis (ESCD), and the Spanish Grupo Español de Investigaci on en Dermatitis de Contacto y Alergia Cutánea. As patch test outcome, reactions between day (D) 3 and D5 were considered.
Beyond these terpene hydroperoxides, BIT 0.1% pet. was the second most common allergen with 4.72% (95% CI: 4.2-5.28%), followed by sodium metabisulfite 1% pet. Conclusions: Despite many positive patch test reactions, it remains controversial whether lin-and lim-OOH should be tested routinely, while at least the two preservatives BIT and sodium metabisulfite appear suitable. The present results are a basis for further discussion and ultimately decision on their implementation into routine testing among the ESCD members.

| INTRODUCTION
The last revision of the European Baseline Series (EBS) was in 2019. 1 Since then, the ESB working group of the European Society of Contact Dermatitis (ESCD) evaluated a group of 11 allergens (haptens) as possible candidates to add to the EBS, because information was yet lacking to definitely include or exclude them according to the contemporaneous criteria. 2 The following substances are considered "audit allergens" not (yet) part of the regular baseline series, which had been selected for evaluation, that is, auditing of their value for routine patch testing: sodium metabisulfite, 2-bromo-2-nitropropane-1,3-diol, diazolidinyl urea, imidazolidinyl urea, Compositae mix II (2.5% or 5% pet), linalool hydroperoxides (lin-OOH), limonene hydroperoxides (lim-OOH), benzisothiazolinone (BIT), octylisothiazolinone, decyl glucoside, and lauryl glucoside. Moreover, Compositae mix, originally suggested to be tested 2.5% pet., was also considered at 5% pet., and Evernia furfuracea (tree moss) was additionally included in the set of audit allergens by some departments. This selection of recommended additions to the EBS for further study had been presented and discussed in Wilkinson et al. 1 A retrospective analysis has since examined the suitability of formaldehyde-releasing preservatives for inclusion in the EBS. 3 Since then, the criteria for inclusion in the EBS have been updated. 4 Specifically, where allergens may cross or coreact it was considered that the lower 95% confidence interval (CI) of the frequency of reactions additional to any existing allergen in the EBS should be above the 0.5% threshold of reactions to be included in the EBS. In addition, as in the case of DMDM hydantoin, where exposure to an allergen is limited and predictable, that allergen may be better placed in a specialized, for example, cosmetic series. For instance, the frequency of reactions to DMDM hydantoin was 3-fold greater than in the baseline series when tested in a cosmetic/facial series. Where results remain equivocal and the 95%CI crosses the 0.5% threshold, it was considered that an allergen should remain as a recommended addition to the EBS for individual countries/ departments to consider adding to their local baseline series.
The present article reports descriptive results offering a broad geographical coverage concerning the above-mentioned scope of audit allergens; results concerning allergens presently included in the EBS will be presented separately. 5 Although the EBS results include the full scope of contributors, data in the present analysis have been contributed by a subset of departments that have patch tested the audit allergens in consecutive patients.

| ESSCA working group of the ESCD
The European Surveillance System on Contact Allergies (ESSCA) is a working group of the ESCD (https://www.escd.org). Its objective is the clinical surveillance of contact allergy. 6,7 To this end, contributing departments (see list of authors and collaborators) submit to the data center in Erlangen either all patch test results, or just patch test results obtained with the EBS (or national or local adaptations thereof), obtained following ESCD standards. 8 This is accompanied by important demographic and clinical information, ranging from "MOAHLFA" characteristics (see Table 1) to a wider range of information according to the ESSCA "minimal data set" definition. 6,7,9 Data from contributing departments are delivered in an anonymous format, or partly, following national network standards, in a pseudonymized format, where the pseudonym cannot be related to actual personal data except in the contributing department itself. This difference is of importance, because only with pseudonymized data can re-investigations of patients be identified and eliminated, to avoid duplication of entries. However, in view of the short study period ranging from, effectively, 1.25 to 2 years, and the special scope of audit allergens considered, the effect of re-consultations appeared negligible. Data were quality checked, providing an "internal report" for each contributing department for scrutiny and approval before pooling of the respective data. 7 Data management and analysis were performed with the R software package (<www.r-project.org>; RRID: SCR_001905), version 4.0.3. For the calculation of 95%CIs to zero proportions an approximation to an exact CI was used. 10 With the objective of presenting a descriptive overview of the morbidity in the patch-tested population, we refrained from employing standardization and adjustment techniques usually necessary in risk-factor and time-trend analyses, respectively.  and data-protection legislation. Clinical data match exactly with those set out in the minimal data set of the ESSCA, which allows them to be exported to other databases with identical categories. Ideally, the centers systematically upload clinical data and the results of patch tests on the day of the last reading, thus providing epidemiologic centralized data in real time. The database has a modular structure that can be used for prospective studies over a definite period of time. For the present study, a specific data form with the "candidate allergens" was added to the original structure of the registry for a 2-year period.

| RESULTS
In total, 21 633 patients were patch tested with the EBS or the TRUE Test baseline series from January 2019 to December 2020 in the participating departments. Because a varying scope of audit allergens has been tested by some of these departments, and in some countries none of these, the individual contribution is lower. Population characteristics according to the MOAHLFA index, 11 extended by the "P-measure" (the proportion of patients positive to at least one allergen from the baseline series 12 ) are illustrated in Table 1 regarding the subset of patients who were patch tested with at least one audit allergen, which is the focus of the present analysis. Results of the GEIDAC obtained with the audit ("candidate") allergens have already been published separately. 13 The audit allergens 1 were tested along with the EBS, that is, in consecutive patients. However, as mentioned, most departments or national groups did not include all audit allergens, and partly not for the entire study period. Hence, the total numbers of single audit allergens tested is markedly smaller and variable, as shown in Table 2. A supplemental analysis stratified for the contributing countries can be found online in Table S1.
Regarding reactivity to Compositae mix II, which was one of the candidate allergens, compared to sesquiterpene lactone (SL) mix, 4005 patients were tested both with SL mix and Compositae mix II 2.5% pet. (SL mix and Compositae mix 5% had not been tested in parallel, thus not permitting a comparison with SL mix). Among these 4005 patients, 19 reacted positive to both, 18 only to Compositae mix II 2.5%, that is, 0.45 (95% CI: 0.27-0.71%), and 18 only to SL mix.
Coupled reactions between decyl and lauryl glucoside were common. In the 4730 patients who were tested with both agents, 37 were positive to both, 45, that is, 0.95 (95% CI: 0.69-1.27%), only to decyl glucoside but not lauryl glucoside, and 14, that is, 0.3 (95% CI: The terpene hydroperoxides, lin-OOHs and lim-OOHs, had been tested in two concentrations each because the optimum patch test concentration has hitherto not been identified, and also to support the interpretation of patch test reactions. Univariate results are presented in Table 2 and co-reactivity in Table 4. Taking the two respective concentrations together, coupled reactivity is moderate, with an OR of 32.6 (95% CI: 25.2-42.1). If, instead, co-reactivity between the two respective lower concentrations is considered in a sensitivity analysis, the OR changes marginally to 28. • Although many patients for whom individual data were available had been tested with all these four fragrances (n = 10 427), 6651 patients had been tested with one or both of the concentrations of lin-OOHs, and 7142 patients had been tested with one or both concentrations of lim-OOHs (see Table 2).
• Among the 4790 tested with all four fragrances of the EBS and any concentration of lin-OOHs, 126 were positive to both, 386 only to at least one fragrance marker, but not lin-OOHs, and vice versa, 234 only to lin-OOHs but not any fragrance marker, the latter amounting to 4.89% (95% CI: 4.29-5.53%) of patients such tested.
T A B L E 1 Demographic and clinical characteristics according to the MOAHLFA index 11 with "P-measure," 12 see note below Note: Note that for the latter cross-tabulation, all three allergen preparations had to be tested in the same patient, whereas in the former two, only the two allergens involved. Patch test results (day 3 to day 5) 2019-2020. OIT and BIT tested in pet.; MI (0.2 and 0.05% pooled) and MCI/MI (0.02 and 0.01% pooled) in aq. Note: In the two rightmost columns and the two bottom rows, resp., results with both concentrations were aggregated, that is, patients positive to one or both concentrations were regarded as positive, and those not reacting to any concentration as negative. Results on clinical relevance of the audit allergens are shown in Table 5 and in more detail concerning product categories involved, online in Table S2. With considerable differences between the allergens, which are difficult to interpret, the share of unknown relevance (summarizing both "not reported" and "unknown relevance") is partly high. The aggregated REIDAC data used, except for Barcelona/ Hospital del Mar contributing via WinAlldat/ESSCA, did not include relevance information; the respective positive cases are not included in the denominator for the two relevance tables. In a few instances relevance information had been documented but could not be related to the product categories used ("not classified"; see Table S2).

T A B L E 4 Coupled reactivity between linalool hydroperoxides (lin-OOH) and limonene hydroperoxides (lim-OOH) in the two concentrations tested
Because ingredient labeling is mandatory only in cosmetics and

| DISCUSSION
This article presents an audit of testing with the recommended additions to the EBS 1 to enable a further revision. With respect to the 0.5% threshold for inclusion, 4 specifically the lower 95% confidence limit to the prevalence estimate, the univariate results in Table 2 are a first approach. Concerning the additional yield beyond a "related" marker/allergen, the calculations added in the text of the results are considered.

| Preservative allergy
With the restrictions on the use of MI it is unsurprising that departments have seen a rapid fall in allergy to both MI and MCI/MI 14,15 back to pre-epidemic levels, or not quite so. 16 Although not permitted in cosmetics, both OIT and BIT were recommended for more routine testing 1 and a statistically significant rise in the prevalence of allergy to BIT, in particular, has been found. 15,17 It seemed probable that this increased prevalence had occurred as a consequence of exposure in domestic cleaning products, detergents, and paints. Notwithstanding co-sensitizations to MI, BIT, and OIT, cross-reactivity might also occur between MI and BIT, and to a greater extent between MI and OIT, 18 the latter also found as a leather-treating agent. 19 Our data confirm labeling terminology and to avoid mis-labelling. 21 Generally, improved ingredient information for medical devices has been called for by the major European scientific societies in the field. 22 Following the withdrawal of MI in leave-on cosmetics, other preservatives must be taking their place, likely including the so-called formaldehyde releasers. These recommended additions to the EBS 1 have been the subject of a separate article and will not be further discussed here. 3,4 However, a nonsignificant upward trend in allergy to sodium metabisulfite has been seen. 17 In 2019, it was felt that there was limited geographic data for this allergen and that relevance was not always apparent. 1 Rubber gloves have in the meantime, by the use of an experimental spot test, been identified as a likely source of sulfites; however, chemical-analytical confirmation is still necessary. 23 In conclusion, with a prevalence of presently 3.0% (95% CI: 2.58-3.51%) and current relevance of 58%, it seems clear that this fairly widespread preservative allergen meets the criteria for inclusion.

| Fragrance allergy
It is recognised that the current fragrance markers in the baseline series are a limited screen to detect contact allergy to fragrance ingredients. One strategy to improve the diagnostic yield has been to test to individual fragrance allergens from the 26 individual fragrance substances for which cosmetic ingredient labeling is mandatory within the European Union. 24,25 Evernia furfuracea (tree moss) has been reported to be potentially of added value beyond the baseline series markers. 24 Extending the actual set of "audit allergens," some departments have consecutively tested with this natural mixture, enabling an assessment of the added value. Although the additional yield from testing E. furfuracea was 0.80% (95% CI: 0.53-1.18%) when compared to FM I, this fell to 0.53% (95%CI: 0.31-0.85%) when compared with colophonium. It seems that this may, therefore, be more a marker of allergy to resin acids as found in colophonium and not warrant further consideration.
Specifically, during production, tree moss is scraped from pine tree bark, leading to contamination and a substantial share of E. furfuracea contact allergy being related to colophonium sensitization. 26 In accordance, 75% of patients who are positive to E. furfuracea in a Danish study have had relevant contact allergy to colophonium. 27 Two other allergens identified as audit allergens were the hydroperoxides of linalool and limonene (lin-OOH and lim-OOH, respectively). 24,25 Although exposure to these seems difficult to confirm in products, 28 clinical studies suggest relevance 29 with positive repeated open application test (ROAT) in those with definite patch test reactions and a smaller proportion (15%) of those even with doubtful patch test reactions. 30 This has led to suggestions that they be included in various national baseline series. 31 There currently remains a dilemma, and a discussion also among the authors of this article, as to whether to include these allergens in the EBS or, as at present, keep them as a recommended addition until a less-irritant alternative is developed.

| Compositae allergy
It has long been recognized that the sesquiterpene lactone (SL) mix is an inadequate screen to diagnose Compositae allergy 33

| Glucosides
In 2017, the North American Contact Dermatitis Group had noticed a steadily increasing frequency of reactions to above 1% of those tested. 36 At the time of the last update of the EBS 1 there were few data from Europe, and hence lauryl and decyl glucoside were included as recommended additions. With a share of positive reactions of 1.73% (95% CI 1.38-2.15%) decyl glucoside appears to be the more frequently reactive of the two. Although the relatively low current relevance of 30.9% is of some concern, this still potentially warrants inclusion in the EBS. Conversely the additional yield from testing lauryl glucoside in addition to decyl glucoside was only 0.30% (95%CI 0.16-0.5%), suggesting it may be better placed in a cosmetic/facial series despite a current relevance of 67.3%. The low additional yield reflects the high frequency of crossreactions between the different glucoside chemicals. 37 In conclusion, we present the results of an audit testing with "recommended additions" to the baseline series 1 with suggestions for updating the EBS (i.e., adding sodium metabisulfite, benzisothiazolinone, and decyl glucoside) and identifying where further work is required (e.g., concerning Compositae mix, and lin-and lim-OOHs). Nicola Wagner: Conceptualization (equal); data curation (equal); investigation (equal); methodology (equal); resources (equal); validation (equal); writingreview and editing (equal). Elke Weisshaar: Conceptualization (equal); data curation (equal); investigation (equal); methodology (equal); resources (equal); validation (equal); writing -