Ear Swelling Test Research Articles

COMPARISON OF MURINE ASSAYS FOR THE IDENTIFICATION OF CHEMICAL SENSITIZERS

Efforts are under way to develop a phenotypic analysis assay capable of identifying and differentiating chemicals with the potential to induce irritation and IgE-mediated and T-cell-mediated sensitization. These studies compare results obtained with the irritancy/phenotypic analysis assay to those obtained in the mouse ear-swelling test (MEST) and the local lymph node assay (LLNA). Female BALB/c mice (5 animals per group) were exposed topically to the human sensitizers 2,4-dinitrochlorobenzene (DNCB) and potassium dichromate (PDC) as well as the irritant methyl salicylate (MSC), following the protocol for each assay. DNCB was identified as a sensitizer in the LLNA and phenotypic analysis assay at concentrations as low 0.25%. In the MEST, DNCB was considered positive in concentrations as low as 0.01 and 0.05% at the 24- and 48-h measurements of the MEST, respectively. PDC tested positive as a sensitizer in the LLNA and the phenotypic analysis assay at concentrations as low as 0.25 and 0.1%, respectively. I...

<I>N</I>-Pyridinyl(alkyl)polyhalogenobenzamides Acting as TNF-α Production Inhibitors

A series of N-pyridinyl(methyl)fluorobenzamides issued from 2,4-dimethyl-6-aminopyridine and 3-aminomethylpyridine were synthesized and evaluated as inhibitors of TNF-α production. Although less active than the corresponding phthalimides, several pentafluorobenzamides exhibited significant activity at 10 μM. N-(4,6-dimethylpyridin-2-yl)pentafluorobenzamide was selected for a preliminary in-vivo assay. Although its inhibitory activity against carrageenan-induced rat paw oedema was moderate, it induced a significant reduction in ear thickness in the PMA-induced mouse ear-swelling test (49± 6% inhibition after a dose of 0.1 mM kg−1, p.o.).

Phototoxicity of quinalphos under sunlight in vitro and in vivo

The phototoxicity of quinalphos (QP) under sunlight was studied by investigating skin sensitization, mouse tail and ear swelling tests and generation of activated oxygen species (AOS). Quinalphos, at an oral dose of 5 mg/kg to the mouse, did not produce any change in water content of the tail, but produced ear swelling under sunlight (60 min). The pesticide did not produce any skin sensitization reaction when applied on depilated skin of guinea pigs (100–500 μg/cm 2) and irradiated under sunlight (60 min). Furthermore, the compound produced a significant amount of AOS such as singlet oxygen ( 1O 2) and superoxide anion radical ( O - 2 ̇ under sunlight. The production of AOS was both concentration (1–20 μg/ml) and irradiation time (0–40 min)-dependent. The generation of AOS was also confirmed by using quenchers of singlet oxygen (sodium azide and DABCO) and superoxide anion radical (SOD).

Contact Sensitivity to Selected Acrylate Compounds in B6C3F1 Mice: Relative Potency, Cross Reactivity, and Comparison of Test Methods

ABSTRACTGiven the increasing prevalence of occupational sensitization to acrylate compounds, n-butyl acrylate (BAC), ethyl acrylate (EAC), and trimethylol propane triacrylate (TMT) were recommended by the National Toxicology Program for hypersensitivity testing in female B6C3F1 mice. The objectives of these studies were to determine the irritating and sensitizing potential of these three compounds using an irritation assay, the murine Local Lymph Node Assay (LLNA), and the Mouse Ear Swelling Test (MEST). The minimal irritating concentration for TMT was determined to be 1.0%, whereas BAC and EAC demonstrated no irritation up to 30%, the highest concentration tested. TMT tested positive in the LLNA at concentrations as low as 0.1% whereas an induction concentration of 0.3% was required to elicit a positive response in the MEST. Furthermore, BAC tested negative in the MEST at induction concentrations as high as 30%, but yielded positive results in the LLNA at concentrations as low as 20%. EAC, at all concentrations tested, was negative in both the MEST and the LLNA. Cross reactivity was only seen when mice were sensitized with TMT and challenged with BAC. In these studies, the LLNA was a more sensitive indicator of the allergic potential of these three acrylates when compared to the MEST.

6-Amino-2,4-lutidine carboxamides: α-aminoamide derivatives as systemic and topical inflammation inhibitors

The development of new potential anti-inflammatory compounds resulting from the incorporation of α-aminoacid residues into 6-amino-2,4-lutidine afforded (N-protected) aminoamides with interesting inhibitory activity. Out of 28 tested compounds, 10 ( 5a, 5b, 7d, 8a, 8b, 8d, 10a, 11b, 12a and 12b) exerted potent (>90%) inhibition in the carrageenan foot edema (CFE) rat model after oral administration of 0.4 mmol kg −1. Except for Cbz-glycyl, Cbz-alanyl, Fmoc-valyl and Cbz-alanyl-glycyl derivatives ( 5a, 5b, 7d and 11b). N-deprotection afforded more active compounds. Introduction of a glycyl residue in the previously studied highly active 3-fluorobenzamide 2, which led to 10a, maintained potent peripheral edema inhibition but had a detrimental effect in the acute TPA-induced mouse ear-swelling model. Glycylglycinamide 12a, which had an ID 50 of 9.0 mg kg −1 in the CFE test, appeared to be the most efficient compound tested in this new series of non-carboxylic nonsteroidal anti-inflammatory drugs. Glycinamide 8a, although less potent in the same assay (14.3 mg kg −1), exerted a significant inhibitory effect in acute and chronic ear-swelling tests after topical application of 3 mg/ear.

Contact Hypersensitivity to Dicyclohexylcarbodiimide and Diisopropylcarbodiimide in Female B6C3F1 Mice

ABSTRACTDicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are two commonly used coupling reagents in protein synthesis resulting in exposure of individuals in chemical and pharmaceutical industries as well as research laboratories involved in protein synthesis and recombinant DNA techniques. The objectives of these studies were to determine the irritation and sensitizing potential of these two compounds when applied topically to B6C3F1 mice. Sensitization potential was assessed by the Mouse Ear Swelling Test (MEST) and the murine Local Lymph Node Assay (LLNA). Concentrations used in the contact hypersensitivity assays were determined by primary irritancy studies. DCC and DIC were identified as both irritants and contact sensitizers with the MEST being a more sensitive indicator of sensitization potential. The MEST identified DCC as a sensitizer at concentrations as low as 0.006% (w/v) 24 hr and 48 hr post challenge and DIC at 0.3% (w/v) and 1.5% (w/v) 24 and 48 hr post challenge, respectively. In the LLNA, the lowest concentrations yielding a significant response were 0.06% (w/v) for DCC and 10% (w/v) for DIC.

Involvement of Substance P in Ultraviolet Irradiation‐induced Inflammation in Rat Skin

The possible involvement of substance P released from primary afferents in rat skin was investigated in cutaneous inflammation following ultraviolet (UV) irradiation. Recordings from c-fibres innervating the UV-exposed hindpaw skin showed long-lasting low-frequency (0.8-1.25 Hz) spontaneous activity. Spontaneously active c-fibres increased to constitute 35.3% of the total population 72 h after UV exposure. Immunohistochemical analysis of substance P-containing nerve fibres in hindpaw skin revealed a significant increase in substance P immunoreactivity 24 h after UV irradiation. Average length of substance P-immunolabelled nerve fibres was about two times higher in UV-exposed compared to control skin. UV-induced oedema was investigated in rat ears using an ear-swelling test. Intradermal injection of either peptide (Spantide) or nonpeptide (CP-96,345) substance P antagonists and epicutaneous application of CP-96,345 reduced UV-induced oedema significantly in the late phase of sunburn (> 12 h after UV exposure). The UV-induced increase in skin blood flow was investigated in hindpaw skin up to 72 h by the laser Doppler technique. Epicutaneous application of CP-96,345 reduced erythema significantly between 12 and 72 h after UV exposure. Thus, our findings suggest the involvement of neurogenic substance P as a proinflammatory mediator in the late phase of UV-induced cutaneous inflammation in rats.

Anti-inflammatory activity of croconazole, a broad-spectrum antimycotic agent, in the arachidonic acid-induced mouse ear swelling test.

Investigations on croconazole, a novel imidazole compound, suggested antiphlogistic properties in vitro. Hence, its anti-inflammatory capacity was tested in vivo using the arachidonic acid-induced mouse ear swelling test, which is a suitable model for screening inhibitors of the lipoxygenase and/or the cyclooxygenase. Topical application of croconazole (1%/0.01%) to the mouse ear induced a maximal inhibition of edema (inhibition: 39%/33%; p = 0.01) which was as strong as the reference nordihydroguaiaretic acid (inhibition: 38.9%; p = 0.01). These results justify further investigations on croconazole to study potential inhibitory effects on proinflammatory arachidonic acid metabolites.

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 D 3 Derivative (Calcipotriol) and Corticosteroids

Vitamin D 3 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 D 3 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, caicipotriol 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 D 3 derivatives are devoided of immunosuppressive effects when applied topically, and (2) that clinical improvement of chronic inflammatory dermatoses observed with topical vitamin D 3 derivatives and corticosteroids is due to different mechanisms.

Evaluation of Several Variations of the Mouse Ear Swelling Test (MEST) for Detection of Weak and Moderate Contact Sensitizers

The ability of a chemical to cause contact sensitization has traditionally been evaluated in animal models typically using the guinea pig. However, these methods are expensive and require subjective analysis of erythema, which makes evaluation of dyes difficult. The mouse ear swelling test (MEST) is a more quantitative and less costly method, but it has not always been reliable for the detection of moderate and weak sensitizers. To identify a MEST that can reliably detect weak sensitizers, several published MEST procedures were examined using the strong sensitizer 2,4-dinitrofluorobenzene (DNFB) and three weaker sensitizers, glutaraldehyde, formalin, and an azo dye (Solvent Red 1 [SR1]). Almost all variations of the MEST procedures detected the strong sensitizer (DNFB) after optimizing the chemical concentration and sensitizing procedure; however, only one protocol detected the weaker sensitizers, glutaraldehyde, formalin, and SR1. This sensitive MEST protocol required test animals to be fed a vitamin A-supplemented diet for 4 weeks prior to sensitization. We concluded that the use of a vitamin A diet with the MEST proved to be the best procedure to enhance the sensitivity and detection of weak sensitizers.

Skin sensitization—A critical review of predictive test methods in animals and man

With the exception of the Draize Test, the guinea-pig test methods currently accepted by regulatory authorities worldwide are well able to predict the potential of a material to cause skin sensitization. Nevertheless, (a) some methods are more sensitive than others (e.g. adjuvant tests are generally more sensitive than non-adjuvant tests); (b) methods cannot be sufficiently standardized to give full reproducibility of results between laboratories; and (c) most methods are based on subjective visual grading of skin reactions—difficulties thus arise when testing coloured or irritant materials. Laboratories must be able to show the sensitivity of the method(s) they use by demonstrating that positive reactions occur with mild/moderate contact allergens rather than the strong/extreme sensitizers currently recommended in certain guidelines, specifically in the EEC Test Method. The sensitivity of the adjuvant tests is such that it is possible to halve the minimum number of animals required by present regulatory guidelines without compromising the capacity of the tests to detect weak/mild sensitizers. A similar review has not yet been made for non-adjuvant tests. Alternative test methods, including some recently developed mouse models, offer several advantages, including more objective endpoints. These tests have not been extensively validated and this precludes their use at present for regulatory purposes other than to confirm the sensitization potential of a material. Two new test methods using mice, the Mouse Ear-swelling Test and the Local Lymph Node Assay, appear promising. They should undergo rigorous interlaboratory testing to determine their sensitivity and specificity. In vitro methods do not represent a viable alternative in the foreseeable future. An approach using quantitative structure-activity relationships is the most likely route to a non-animal model, but this will require considerable research, development and validation. Human sensitization tests have generally not been used for the classification of substances as non-sensitizers. This is because of an absence of internationally agreed test protocols, the lack of positive controls and because the methods for establishing the sensitivity of human tests are less developed than for animal tests. Nevertheless, for products for which direct human contact is intended, predictive tests in human volunteers can be considered. The EEC Directive for the Classification, Packaging and Labelling of Dangerous Substances provides a reasonable approach to the evaluation of skin sensitizers. In contrast, one of the options to meet the requirements of the EEC Dangerous Preparations Directive that preparations containing ⩾ 1% of the sensitizer should be labelled, fails to take into account the potency of different sensitizers and the fact that incorporation of weak sensitizers into formulations at a concentration ⩾ 1% may not constitute a sensitizing hazard.

Variability of a Mouse Ear Swelling Test (MEST) in Predicting Weak and Moderate Contact Sensitization

Variability of a Mouse Ear Swelling Test (MEST) in Predicting Weak and Moderate Contact Sensitization. DUNN, B. J., RUSCH, G. M., SIGUN, J. C, AND BLASZCAK, D. L. (1990). Fundam. Appl. Toxicol. 15, 242–248. An investigation was conducted at two independent laboratories for the purpose of evaluating the ability of a mouse ear swelling test (MEST), previously developed and validated by S. C. Gad, B. J. Dunn, D. W. Dobbs, C. Reilly, and R. D. Walsh (1986, Toxicol. Appl. Pharmacol. 84, 93–114), to predict contact sensitization induced by weak to moderate contact sensitizers. Twenty-six coded chemicals, 23 with weak to strong potential to induce sensitization in animals and/or humans and 3 negative controls, were investigated. Each laboratory tested 18 materials: 10 chemicals were common to both labs and 16 chemicals were divided so that 8 were unique to each of the two labs. The methods and chemicals utilized were the same as those used and reported by Gad el at. (1986) and, therefore, data generated from this investigation were compared to results from their research. Contrary to results reported by Gad and co-workers, findings from the present study suggest that this MEST is a useful model for identifying strong contact sensitizers, but is not reliable for detecting weak to moderate allergens.

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice

Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 μg to 1000 μg. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.

Immune response to BCG-Moreau (Rio de Janeiro) strain. Spectrum of delayed hypersensitivity in genetically defined mice.

Generation of delayed hypersensitivity (DTH) in genetically defined mice immunized with Mycobacterium bovis BCG of the Moreau (Rio de Janeiro) strain was studied. This vaccine strain has been reported as the most virulent and able to induce strong tuberculin sensitivity. Mice were selected by the expression of Bcg gene trait, by responsiveness to mycobacterial antigens and H2 haplotype. DTH was evaluated by the ear-swelling test of mice immunized subcutaneously with live BCG at doses ranging from 1 microgram to 1000 micrograms. A survey of inbred strains of mice showed H2b and H2q mice as high responders, H2d as an intermediate responder, H2k as a low responder and H2a as a non-responder. Study of H2-congenic pairs of high and non-responder strains showed significant DTH in all mice independently of the genetic background and H2 haplotype. A mouse strain expressing Bcg (r) trait displayed DTH superior to a Bcg (s) strain. Comparison of DTH response of strains expressing Bcg (r) or (s) trait showed no relationship between the Bcg locus and DTH to mycobacteria. These data suggest DTH is under polygenic control including the major histocompatibility complex but excluding the Bcg locus.

Changes in cellular immunity and nutritional status in mice after thermal injury

A 13 per cent body surface area (BSA), full skin thickness burn was inflicted on LACA male mice and the changes in cellular immunity and nutritional status were observed. The results showed that thymus, spleen and circulating lymphocytes were significantly involved. A diminished mitogen responsiveness of spleen cells and altered peritoneal macrophage function were confirmed. Ear swelling tests indicated that the cellular immunity of burned mice was most severely depressed in week 2 postburn. The present study also showed that the dramatic change in nutritional status occurred earlier than that in cellular immunity and suggested the importance of early nutritional support after thermal injury.

Comparison of a Radioisotopic Incorporation method and the Mouse Ear Swelling Test (MEST) for Contact Sensitivity to Weak Sensitizers

Comparison of a Radioisotopic Incorporation method and the Mouse Ear Swelling Test (MEST) for Contact Sensitivity to Weak Sensitizers

An animal model for nonimmunologic contact urticaria

The suitability of the guinea pig for studies on nonimmunologic contact urticaria (NICU) was investigated. Groups of five, female Hartley guinea pigs were challenged by applying 50 μl of various concentrations of human NICU agents, benzoic acid (BA), sorbic acid (SA), cinnamic acid (CA), cinnamaldehyde (CAL), methyl nicotinate (MN) and dimethyl sulfoxide (DMSO), in absolute ethyl alcohol to both sides of the earlobe. The thickness of the ear (0.85 ± 0.11 mm, x ± SD, 100 animals) was measured with a string micrometer before application and at 5- to 30-min intervals thereafter. Maximal increase in ear thickness was produced within 30 to 40 min by 20% BA (0.70 ± 0.09 mm), 10% SA (0.30 ± 0.05 mm), 15% CA (0.58 ± 0.12 mm), 5.0% CAL (0.37 ± 0.09 mm), 0.2% MN (0.80 ± 0.03 mm), and 100% DMSO (0.74 ± 0.11 mm). All responses were dose dependent. The histology of the maximal reactions showed dermal edema, and intra- and perivascular infiltrate of heterophils (neutrophils in man), eosinophils, and some lymphocytes. DMSO also caused vacuolization of the epidermal cells. CAL, MN, and DMSO, applied to different parts of the guinea pig body, produced only erythema most frequently on the upper back, but BA, SA, and CA did not cause any reactions. The guinea pig ear-swelling test provides a quantitative animal model to screen human NICU agents.