Classification of contact allergens according to potency: proposals.

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of potency and categorisation. Included here are detailed and specific recommendations for how best the results of the three test methods considered can be used for the categorisation of chemical allergens as a function of skin sensitisation potency.

Skin sensitisation testing--new perspectives and recommendations.

Various methodological aspects of skin sensitisation testing have been explored, particularly in the context of animal welfare considerations and reliability and sensitivity of test methods. Recommendations are made for the conduct of current and proposed OECD skin sensitisation tests with respect to appropriate test configurations for the purposes of hazard identification and labelling, and the requirement for positive controls. Specifically, the following aspects of guinea pig sensitisation test methods have been addressed: (1) the number of test and control animals required; (2) the option of using joint positive controls between independent laboratories; (3) the choice of positive control chemicals; (4) the optimal conduct and interpretation of rechallenge; and (5) the requirement for pretreatment with sodium lauryl sulfate. In addition, the use of the murine local lymph node assay (LLNA) has been considered. A number of conclusions have been drawn and recommendations made as follows: In many instances, particularly with the conduct of the guinea pig maximisation test, it is acceptable to halve the number of test and control animals used. An optional scheme for the conduct of joint positive control studies within a co-ordinated group of laboratories is appropriate. Only one positive control chemical (alpha-hexyl cinnamic aldehyde) is necessary for the routine assessment of assay sensitivity. The proper conduct and interpretation of rechallenge can provide valuable information and confirmation of results in guinea pig sensitisation tests. Sodium lauryl sulfate should no longer be used as a pretreatment in the guinea pig maximisation test. The LLNA is a viable and complete alternative to traditional guinea pig test methods for the purposes of skin sensitisation hazard identification. These recommendations provide the opportunity for both animal welfare benefits and improved hazard identification.

Skin sensitization testing in potency and risk assessment.

The purpose of this article is to review, and make recommendations for, the use of relevant skin sensitization test methods, for the purposes of determination of relative potency and the threshold dose necessary for the induction of skin sensitization, and for risk assessment. In addressing the first area, the utility of three guinea pig tests (the guinea pig maximization test, the occluded patch test, and the open epicutaneous test) of the local lymph node assay (LLNA) and of human volunteer testing for the assessment of relative potency and identification of thresholds for sensitization were considered. The following conclusions were drawn. (1) Although attempts have been made to modify the guinea pig maximization test for the purposes of deriving dose-response relationships, this method is usually unsuitable for determination of relative sensitizing potency. (2) Guinea pig methods that do not require the use of adjuvant and which employ a relevant route of exposure (the occluded patch test and the open epicutaneous test) are more appropriate for the assessment of relative skin-sensitizing potency. (3) The LLNA is suitable for the determination of relative skin sensitizing potency, and the adaptation of this method for derivation of comparative criteria such as EC3 values (the estimated concentration of test chemical required to induce a stimulation index of 3 in the LLNA) provides an effective and quantitative basis for such measurements. (4) For all the methods identified above, potency is assessed relative to other chemical allergens of known skin sensitizing potential. The estimation of likely threshold concentrations is dependent upon the availability of suitable benchmark chemicals of known potency for human sensitization. (5) Human testing (and specifically, the Human Repeat Insult Patch Test) can provide information of value in confirming the absence of skin sensitizing activity of formulations and products under specific conditions of use and exposure. Based on the above, the following recommendations are made. (1) If results are already available from suitable guinea pig tests, then judicious interpretation of the data may provide information of value in assessing relative skin sensitizing potency. This option should be explored before other analyses are conducted. (2) The LLNA is the recommended method for new assessments of relative potency, and/or for the investigation of the influence of vehicle or formulation on skin sensitizing potency. (3) Whenever available, human skin sensitization data should be incorporated into an assessment of relative potency. With respect to risk assessment, the conclusion drawn is that all the available data on skin-sensitizing activity in animals and man should be integrated into the risk-assessment process. Appropriate interpretation of existing data from suitable guinea pig studies can provide valuable information with respect to potency, as the first step in the development of a risk assessment. However, for de novo investigations, the LLNA is the method favored for providing quantitative estimations of skin-sensitizing potency that are best suited to the risk assessment process. Finally, human testing is of value in the risk assessment process, but is performed only for the purposes of confirming product safety.

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation.

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.

Suboptimal non-inflammatory concentrations of haptens may elicit a contact sensitivity reaction when used as a mix.

Contact dermatitis is a cutaneous inflammatory reaction mediated by hapten-specific T cells. We used a murine model to investigate the contact sensitivity reaction elicited by different concentrations (optimal and suboptimal) of the haptens DNFB and oxazolone applied singly or in combination. The simultaneous application of DNFB and oxazolone at optimal concentrations (0.2% and 0.4% respectively) did not significantly increase the ear swelling response induced by each of the allergens when applied singly. No contact sensitivity response was observed when the haptens were tested individually at subthreshold concentrations (0.05% and 0.1% respectively). However, mixing the 2 molecules at the same concentrations gave rise to a clinical contact sensitivity reaction. The simultaneous application of the haptens at a 2 x higher, but still suboptimal concentrations (0.1% and 0.2% respectively), elicited an inflammatory response that was significantly greater than the responses elicited by either of the haptens when applied separately. These results suggest that a "false-positive" reaction to a mix may reveal a genuine sensitization to the constituents.

Major histocompatibility complex class I-restricted CD8+ T cells and class II-restricted CD4+ T cells, respectively, mediate and regulate contact sensitivity to dinitrofluorobenzene.

Contact sensitivity (CS) is a form of delayed-type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed-type hypersensitivity responses, by CD4+ T helper-1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8+, CD4+, or both, T cells. MHC class I single-deficient and MHC class I/class II double-deficient mice, both of which have a drastic reduction in the number of CD8+ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II-deficient mice and normal mice treated with an anti-CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti-CD8 mAb depletion of class II-deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4+ T cells in class II-deficient mice by anti-CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I-restricted CD8+ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II-restricted CD4+ T cells down-regulate this inflammatory response.

In vitro T cell response to staphylococcal enterotoxin B superantigen in chronic plaque type psoriasis.

Recent studies have demonstrated the important role of CD4+ T cells in the pathophysiology of psoriasis. One of the current hypotheses is that triggering of the psoriatic inflammatory process could be secondary to CD4+ T cell activation by bacterial superantigens in the skin. In this study, IL-2-derived T cell lines were recovered from the blood and the skin of 4 patients with chronic plaque type psoriasis and of 2 patients with allergic contact dermatitis (ACD). Blood and skin T cell lines were tested for their ability to proliferate in vitro to staphylococcal enterotoxin B (SEB) presented by MHC class II expressing antigen-presenting cells. The results showed a significantly higher SEB-induced T cell proliferation in skin T cell lines as compared to blood T cell lines in 3 out of 4 psoriatic patients and in one of the 2 ACD patients. No difference between the skin and blood T cells for their response to phytohemagglutinin was observed. Furthermore the blood T cell lines from both patients and control individuals responded equally well to SEB. Thus psoriatic skin T cell lines were characterized by an enrichment in SEB-responding T cells. Since similar enhancement of SEB-responsive T cells was occasionally found in ACD patients, we propose that SEB could be an environmental factor associated with rather than responsible for psoriatic inflammation.

Establishment of nickel-specific T cell lines from patients with allergic contact dermatitis: comparison of different protocols.

Allergic contact dermatitis is a delayed-type hypersensitivity reaction, secondary to hapten-specific T lymphocyte activation in sensitized individuals. The present study reports on the establishment of T cell lines from peripheral blood mononuclear cells (PBMC) of nickel-allergic patients, initially cultured with nickel, IL-2, or PHA and IL-2. It was possible to derive hapten-specific T cell lines from the three protocols, and the best proliferative responses to nickel were observed when PBMC were cultured in the presence of nickel in vitro. T cell lines initially cultured with IL-2 always gave better specific proliferative responses to nickel than those derived with PHA and IL-2. Phenotypical analysis of the nickel-specific T cell lines showed that they were mainly composed of activated CD8+ TcR alpha beta + T lymphocytes. These results emphasize the importance of initial culture conditions for the generation of hapten-specific T cell lines and suggest that CD8+ lymphocytes could play an important role in the pathogenesis of allergic contact dermatitis.

Optimization of the mouse ear swelling test for in vivo and in vitro studies of weak contact sensitizers.

Murine models for the assessment of the contact sensitizing properties of chemicals rely on mouse ear swelling tests (Mest), which are not sensitive enough to detect weak sensitizers. The aim of the present study was to develop in mice an adjuvant-free Mest appropriate for in vivo detection of any type of sensitizer (weak to strong), and useful for in vitro assessment of contact sensitivity (CS). 3 haptens were tested: dinitrochlorobenzene (DNCB), para-phenylenediamine (pPD) and isoeugenol. We compared various protocols for induction of the CS reaction, differing by the site of induction, the number of applications and the concentrations of the 3 haptens. Comparison of the induction site for optimal CS reaction showed that, in Balb/c mice, the back was a better site of induction than the abdomen. Detection of the sensitizing properties of weak sensitizers (pPD, isoeugenol) was possible using an adjuvant-free protocol, provided that the induction phase comprised hapten applications on 3 consecutive days on the backs of animals. For DNCB, one application was sufficient to obtain optimal CS reaction. For all 3 haptens, a secondary response in vitro was obtained using semi-purified lymph node T cells from animals sensitized 5 days before with the optimized Mest. These results demonstrate that the Mest could be a useful experimental model for the study of all types of contact sensitizers.

Contact sensitivity in mice: differential effect of vitamin D3 derivative (calcipotriol) and corticosteroids.

Vitamin D3 derivatives are new compounds used topically for the treatment of psoriasis. To get better insights into the mechanisms of action of these compounds, we studied the effect of local treatment with calcipotriol (vitamin D3 synthetic analogue) and compared it to that of betamethasone dipropionate in a murine contact sensitivity (CS) test, the Mouse Ear Swelling Test. Two haptens were used: oxazolone and paraphenylenediamine. Betamethasone and calcipotriol exerted a differential effect on the delayed-type hypersensitivity response. When drugs were applied to the abdomen (sensitization site) before sensitization, no effect was observed. When betamethasone was applied to the abdomen for 4 consecutive days after epicutaneous sensitization, a diminution of the CS response to the relevant hapten was observed, whereas calcipotriol given in the same conditions did not affect the reaction. Ointments were then administered to the ear (elicitation site) either for 4 consecutive days prior to the challenge, or for 4 days before and 2 days after the challenge. In both conditions, calcipotriol and betamethasone exerted a differential effect on elicitation, the latter inhibiting and the former increasing the CS response to oxazolone and paraphenylenediamine. From these results we conclude: (1) that vitamin D3 derivatives are devoided of immunosuppressive effects when applied topically, and (2) that clinical improvement of chronic inflammatory dermatoses observed with topical vitamin D3 derivatives and corticosteroids is due to different mechanisms.

[Smoking among students of the CM2) (2d-year intermediate course): evaluation of knowledge and behavior]. / Tabagisme chez des élèves de CM2: évaluation des connaissances et des comportements.

This paper reports the results of a survey conducted on smoking among 1490 eleven years old schoolchildren. They answered to an individual questionnaire before and after they had been informed on smoking by school doctors. In this age group, 26.2% of the children have already had some contact with cigarette, and 0.5% smoke regularly. The household smoking habits have an important influence on the attitude of children toward smoking. Knowledges on smoking dangers have been evaluated before and after information. This survey shows the importance of smoking prevention, especially among young children.

Dissociation of allergenic and immunogenic functions in contact sensitivity to para-phenylenediamine.

Contact sensitivity to para-phenylenediamine (PPD) is a frequent delayed-type hypersensitivity resulting in contact dermatitis. The aim of the present study, conducted in 16 patients allergic to PPD (as assessed by a positive patch test), was to get better insight into the mechanism of T-cell activation in PPD contact sensitivity. PPD was unable to induce significant proliferation of T cells from a first set of 9 patients. In 7 further patients, lymphocyte proliferation was assessed using PPD and 2 PPD metabolites, namely Brandrowski's base (BB) and benzoquinone (BQ). BB specifically stimulated T-cell proliferation in a dose-dependent fashion in all 7 patients whereas BQ, like PPD, was ineffective. The peripheral blood mononuclear cells (PBMC) of 8 PPD nonallergic individuals did not respond to either PPD, BB or BQ. We concluded from this study that: (1) the immunogenic hapten in PPD hypersensitivity is not PPD itself, and (2) BB might be the oxidative derivative of PPD endowed with T-cell-activating properties. Further support to this statement was provided by the observation that a T cell line derived from PBMC of a PPD-allergic patient in the presence of PPD responded to BB but not to PPD. Our in vitro results suggest that PPD is a prohapten which when applied on the skin is metabolized and converted into products (such as BB) which are the immunogenic haptens able to activate specific T cells.