Monitoring levels of preservative sensitivity in Europe. A 10-year overview (1991-2000).

A 10-year multicentre analysis of the frequency of sensitivity to common preservatives collected in 16 centres in 11 countries has shown stable but persisting high levels of sensitivity to formaldehyde and 5-chloro-2-methyl-4-isothiazolin-3-one + 2-methyl-4-isothiazolin-3-one (MCI/MI). It has also revealed a significant increase in the level of reactivity to methyldibromoglutaronitrile (MDBGN) from 0.7% in 1991 to 3.5% in 2000. The current high level of sensitivity to MDBGN requires an urgent safety re-evaluation and risk assessment update along with consideration of immediate lowering of use concentrations, especially in leave-on products.

Comparison of the sensitivities of the Buehler test and the guinea pig maximization test for predictive testing of contact allergy.

International test guidelines, such as the Organisation for Economic Cooperation and Development (OECD) guideline #406, recommend 2 guinea pig methods for testing of the contact allergenic potential of chemicals: the Guinea Pig Maximization Test (GPMT) and the Buehler test. Previous comparisons between the methods suggested that the Buehler test was less sensitive than the GPMT although modified Buehler test protocols were used. Parallel GPMT and Buehler tests were conducted according to OECD guideline #406 using a multiple-dose design and test results were analysed using a standard logistic dose-response model. To compare the sensitivity of the 2 test procedures the test conditions were kept identical and the following chemicals with a range of sensitization potentials were tested: chloraniline, chlorhexidine, eugenol, formaldehyde, mercaptobenzothiazole and neomycin sulphate. Formaldehyde and neomycin sulphate were strong sensitizers in both tests. Mercaptobenzothiazole, eugenol and chloraniline were all strong sensitizers in the GPMT, eugenol and mercaptobenzothiazole were negative in the Buehler test and equivocal results were obtained with chloraniline. Chlorhexidine was negative in the GPMT and equivocal responses were obtained with the Buehler test. Higher induction concentrations were needed to show allergenicity in the Buehler test and for some allergens the Buehler test protocol was not sensitive enough to demonstrate allergenic potential.

Cross-reactivity patterns of cobalt and nickel studied with repeated open applications (ROATS) to the skin of guinea pigs.

BACKGROUND: The relevance of patch-test reactivity to chemicals on cross-challenge is hard to state, but it is generally assumed that the patient might risk a relapse of contact dermatitis when exposed to the cross-reacting compound(s). OBJECTIVE: To study relevance by using the repeated open application test (ROAT) and applying the inducing allergen cobalt chloride (CoCl(2)) or nickel sulfate (NiSO(4)) as well as the possibly cross-reacting compound (NiSO(4) or CoCl(2)) topically to guinea pigs. METHOD: Animals were induced according to the guinea pig maximization test (GPMT) method, patch tested and then treated for 10 days using ROATs. Sensitivity thresholds were determined with serial dilution tests. RESULTS: Guinea pigs induced with CoCl(2) reacted in patch testing (100%) and in ROATs to CoCl(2) (93%) but not to NiSO(4). Animals induced with NiSO(4) reacted in patch testing to NiSO(4) (100%) but not to CoCl(2), and in the ROATs to NiSO(4) (41%) and less to CoCl(2). CONCLUSIONS: Our results support the assumption that the concomitant patch test reactivity is due to multiple sensitizations rather than cross-reactivity. We previously found that animals induced with palladium chloride (PdCl(2)) also reacted to NiSO(4) on patch testing but not in the ROATs, indicating that the results from patch testing might overestimate the risk of a relapse. ROATs in patients with solitary and/or concomitant sensitivity to CoCl(2), NiSO(4) or PdCl(2) are desirable.

Cross-reactivity patterns of palladium and nickel studied by repeated open applications (ROATs) to the skin of guinea pigs.

Cross-reactivity is usually studied with patch test techniques, but the relevance of a single 1-2 day exposure under occlusion can be questioned. To study relevance, animals were induced with PdCl2 or NiSO4 according to the guinea pig maximization test method and then treated for 10 days according to the repeated open application test (ROAT) method. Animals induced with PdCl2 reacted in the ROATs to PdCl2 (100%) but rarely to NiSO4. Animals induced with NiSO4 reacted in ROATs to the same degree with NiSO4 and PdCl2 (23-30%). The concordance between pre-ROAT patch test results and ROAT outcome was high for PdCl2 (100%) and low (10-40%) for NiSO4. Patch testing seems to overestimate the risk of skin reactions when guinea pigs sensitive to PdCl2 are treated topically with NiSO4. The finding from patch test studies that animals induced with NiSO4 react only to NiSO4 but not to PdCl2 was not confirmed. Repeated open applications more adequately mimic exposure conditions than does patch testing.

Skin sensitization risk assessment: a comparative evaluation of 3 isothiazolinone biocides.

Many of the chemicals in common use possess, to some degree, a capacity to cause skin sensitization. Consequently, it is important to conduct a thorough and accurate risk assessment when it can be anticipated that such chemicals are likely to come into contact with human skin. Such risk assessments must consider the nature and extent of dermal exposure together with the sensitizing potency of the chemical. Whilst the exposure elements of the risk assessment process are often well understood, or can at least be reasonably predicted on the basis of the proposed usage of the chemical, the determination of skin sensitizing potency may be problematic. In this paper, we describe an approach to the determination of relative potency of 3 isothiazolinone biocides. Initially, the local lymph node assay (LLNA) provides an estimate of relative sensitizing potency. Where appropriate, this can be followed by confirmatory testing in the human repeat insult patch test (HRIPT). The data generated reveal that (chloro)methyl isothiazolinone (CMI) is a significantly stronger sensitizer than either methyl trimethylene isothiazolinone (MTI) or benzisothiazolinone (BIT). Furthermore, both the LLNA and the HRIPT data show consistently that BIT is a somewhat weaker skin sensitizer than MTI. As an important additional component of the safety evaluation, the ability of these sensitizing isothiazolinones to cross-react has been considered; the data indicate that the more weakly sensitizing isothiazolinones do not elicit allergic reactions in subjects sensitized to MCI. The implications for use of these biocides in various product types are discussed.

Routine patch testing with the sesquiterpene lactone mix in Europe: a 2-year experience. A multicentre study of the EECDRG.

To test the screening value of the sesquiterpene lactone (SL) mix in Europe today and describe epidemiological and clinical features of Compositae-sensitive patients, the SL mix 0.1% pet. was included in the standard patch test series in 11 European clinics. 10,695 patients were tested, and 106 (1%) had positive reactions, 74% of which were of current or old relevance. The prevalence of positive reactions varied between 0.1 and 2.7% in different centres. The median age of the 66 females was 51.5 years, and 55.2 in the 40 males. The 20 occupationally sensitized had a higher % of males and a median age of 43 years, whereas 7 UV-sensitive patients had a median age of 72 years. Garden plants were the major suspected sensitizers and the clinical patterns were partly, in accordance with plant dermatoses in general, involving hands, forearms and face, and partly widespread eczema in a large proportion of the patients. More than 1/3 were positive to perfume and/or colophony, possibly reflecting cross-reactivity. With only 1 case of active sensitization and no irritant reactions, the SL mix is a safe allergen and the overall prevalence of positive reactions supports its continued use in the standard patch test series.

Sensitizing potential in mice, guinea pig and man of the preservative Euxyl K 400 and its ingredient methyldibromo glutaronitrile.

The allergenicity of the preservative Euxyl K 400 and its principal allergen methyldibromo glutaronitrile (MDBGN) (1,2-dibromo-2,4-dicyanobutane) was investigated using 3 animal models; in mice, the local lymph node assay (LLNA) and in guinea pigs, the guinea pig maximization test (GPMT) and the cumulative contact enhancement test (CCET) with a dose-response protocol included. Previous attempts to define the sensitization capacity of these chemicals have given conflicting results. For comparison, the frequency and causes of positive patch test reactions to Euxyl K 400 and MDBGN were studied in patients referred to an occupational dermatology clinic. This investigation showed that Euxyl K 400 and MDBGN can give rise to contact allergy in man and that the relevant cases found mainly had similar exposure as non-occupational cases. A contact allergenic potential could be detected for MDBGN in 2 animal models, i.e., the CCET and the LLNA, and also for Euxyl K 400 in the LLNA. However, statistical analysis of the results from the GPMT with MDBGN failed to detect the sensitizing potential of this particular allergen. The results indicate that to be able to detect the allergenic potential of Euxyl K 400 and MDBGN, a predictive test method with multiple topical applications at induction is required. It is therefore important that an investigator is aware of the possibility of using various predictive test models for investigation of potential contact allergens.

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.

Free radicals as potential mediators of metal-allergy: Ni2+- and Co2+-mediated free radical generation.

The generation of free radicals by Ni(2+) and Co(2+) was studied at physiological pH in H(2)O(2)-containing solutions in the absence and presence of various radical-mediating ligands and in human peripheral blood mononuclear cell (PBMC) cultures. With ESR spectroscopy, free radical species were identified and quantitated by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Co(2+) generated hydroxyl radicals from H(2)O(2) in PBS solutions containing glutathione (GSH) or histidine (His). Omission of GSH or His from the reaction mixture significantly reduced the ESR-signal, indicating the importance of metal-chelation in free radical generation. Carnosine did not significantly enhance the reactivity of Co(2+) toward H(2)O(2), whereas cysteine (Cys) and N-acetylcysteine (NAC) suppressed free radical generation. Under identical reaction conditions, Ni(2+) was markedly less reactive toward H(2)O(2) in comparison with Co(2+). GSH, His, Cys and NAC did not enhance free radical generation of Ni(2+) from H(2)O(2). However, in the presence of carnosine weak but significantly enhanced ESR intensities were found. Incubation of PBMC cultures from healthy subjects with Co(2+) (10-50 microM) yielded the DMPO-.OH adduct, suggesting Co(2+)-mediated hydroxyl radical generation. In contrast, incubation of PBMC cultures with Ni(2+) (10-50 microM) did not produce a detectable ESR-signal. Ascorbic acid efficiently inhibited Co(2+)-mediated free radical generation in PBS solutions and PBMC cultures. The observed difference in free radical generating capacity between Ni(2+) and Co(2+) is of interest with respect to the absence of cross-reactivity between the two metal-ions in experimental allergic contact dermatitis.

Speciation of chromium in tanned leather gloves and relapse of chromium allergy from tanned leather samples.

There are concerns that wearing chromium tanned leather gloves can provoke a lapse of hand dermatitis in chromium sensitive patients. Various forms of chromium have different biological effects and the determination of total chromium (both soluble and insoluble), which often is used in clinical studies, may therefore not be an adequate measure of the adverse exposure. However, investigations, where dissolution of hexavalent chromium from leather gloves has been experimentally studied, could not been found in the literature. The aim of this pilot study was therefore to determine the total amount of chromium and the amount of sweat leachable chromium in different leather glove materials and also to apply samples of these materials in a patch test to chromium sensitive patients to see if they could provoke a reaction. The results showed tha chromium tanned leather contained about 3% (m/m) chromium. Chromium can also be found in dyed vegetable tanned leather, probably emanating from the leather dye. Small amounts (average 0.08% m/m) of leachable hexavalent chromium were found in both chromium and vegetable tanned leathers. Thus, the risk of relapse of chromium dermatitis cannot be disregarded and further studies on the causes of relapse of chromium dermatitis are necessary.

Lack of antagonism to Ni2+ and Co2+ contact allergy from other essential divalent metal ions.

The potential antagonistic effects of Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ on contact allergy to Co2+ and Ni2+ were studied. The immune response was characterized by the Co2+ or Ni2+ mediated cellular [methyl-3H]thymidine uptake in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects contact-allergic to Co2+ and Ni2+ and 6 non-allergic control individuals. Results from the in vitro experiments were further evaluated with Co2+-sensitized guinea pigs according to the modified Freund's complete adjuvant test. Ni2+ and Co2+ (10-50 microM) significantly increased the lymphocyte proliferation in PBMC cultures from contact-allergic subjects in comparison with those from control individuals. Pretreatment of the PBMCs with Ca2+, Fe2+, Mg2+ (10-100 microM) or Mn2+ (1-10 microM) did not influence, while Zn2+ (100 microM) enhanced, and Cu2+ (5 and 10 microM) markedly reduced the Ni2+ and Co2+ mediated cellular [methyl-3H]thymidine uptake. The inhibition of the Ni2+- and Co2+-induced cell proliferation by Cu2+ in vitro was probably related to toxicity, since the viability of the cells was significantly reduced by applied combinations of Ni2+ or Co2+ with Cu2+. Topical pretreatment of Co2+-sensitized guinea pigs with maximum non-irritating doses of CuCl2 x 2H2O (0.8%) did not affect the challenge testing to CoCl2 x 6H2O (0.1 and 0.3%). In conclusion, our combined in vitro and in vivo results indicate that Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ and Zn2+ are not able to antagonise the formation of Ni2+ and Co2+ antigens.

Free radicals as potential mediators of metal allergy: effect of ascorbic acid on lymphocyte proliferation and IFN-gamma production in contact allergy to Ni2+ and Co2+.

A possible free radical mechanism in metal allergy was investigated in peripheral blood mononuclear cell (PBMC) cultures from 6 subjects, contact allergic to Ni2+ and Co2+, and 6 control individuals. Ni2+ and Co(2+)-mediated free radical generation was studied with electron spin resonance spectroscopy. The immune response was characterized by cellular [methyl-3H]thymidine uptake and interferon-gamma (IFN-gamma) production Ni2+ and Co2+ (10-50 microM) significantly increased lymphocyte proliferation and IFN-gamma production in PBMC cultures from contact allergic subjects in comparison with cultures from controls. Inhibition of Co(2+)-mediated free radical generation by ascorbic acid did not influence cellular [methyl-3H]thymidine uptake and IFN production. Detectable amounts of free radicals were not obtained with Ni2+. We therefore conclude that it is unlikely that free radicals are involved in contact allergy to Ni2+ and Co2+.

Quantification and specificity of the repeated open application test (ROAT). A methodological study using cobalt and colophony in guinea pigs.

The repeated open application test is used to assess the clinical relevance of positive patch test reactions to ingredients of formulated products. The great variation in outcome is usually claimed to be related to the concentration of the allergen responsible. We have here studied the quantitative aspects, specificity and effect of patch testing on the outcome of the repeated open application test in an animal model, using guinea pigs sensitized with cobalt chloride or colophony. Thresholds of sensitivity were determined before and after the topical treatments. Clear dose-response relationships were established. The reactivity in sham-treated controls and to the vehicles was minimal. The concordance between patch test results and outcome of the use tests was concentration-dependent and at low concentrations < 50%. The repeated open application test is a useful method, but some of the basic issues need further evaluation. This animal model will hopefully serve this purpose.