Mouse Ear Swelling Test Research Articles

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.

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.

Prevention of contact hypersensitivity to topically applied drugs by ethacrynic acid: potential application to transdermal drug delivery

Transdermal drug delivery systems have many advantages. However, the extension of this technology to additional drugs is limited by the development of contact sensitization to topically applied drugs. Ion channel modulators have been reported to inhibit elicitation of allergic contact sensitivity. We have developed a mouse ear swelling test (MEST) for sensitization to topically applied nadolol, albuterol, clonidine, and chlorpheniramine. Pre-treatment of the backs of mice with ethacrynic acid (0.5% w/v), prevents subsequent sensitization from topical application of the above drugs. Ethacrynic acid must be applied prior to the sensitizing drug, and the effects of ethacrynic acid are specific for the site of application. Skin permeation studies determined that reduction in sensitization was not the result of reduction in drug penetration. Ethacrynic acid-induced inhibition does not induce a generalized immunosuppression as the mice can be sensitized to drugs or haptens applied to other sites. It is proposed that counter-sensitizers can be incorporated into transdermal drug delivery systems to prevent sensitization, and expand the application of this technology to potentially sensitizing drugs.

Dietary vitamin A enhances sensitivity of the local lymph node assay

Murine assays such as the mouse ear swelling test (MEST) and the local lymph node assay (LLNA) are popular alternatives to guinea pig models for the identification of contact sensitizers, yet there has been concern over the effectiveness of these assays to detect weak and moderate sensitizers. Much work has been done to improve the sensitivity of the MEST, including the addition of a vitamin A acetate (VAA) enriched diet, which increases its sensitivity. Vitamin A acetate has been reported to increase the numbers of Langerhans cells (antigen presenting cells) in the skin, which could in turn enhance the cellular immune response. Because the LLNA relies on tritiated-thymidine incorporation by proliferating T cells during the induction phase, we have studied the potential of the VAA diet to enhance sensitivity of the LLNA. Results indicate that the VAA enriched diet significantly increased the LLNA sensitivity to formalin, eugenol, glutaraldehyde, trimellitic anhydride, and an azo dye at concentrations where no proliferation was observed in mice maintained on the standard diet. Maintenance on a VAA diet for 3 weeks prior to initiating the sensitization procedure was optimal. Thus, incorporation of a VAA diet improves the sensitivity of the LLNA as a quick, objective, and relatively inexpensive screen for detecting moderate and weak contact sensitizers.

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.

Classification of chemicals as sensitisers based on new test methods

For the last decade, classification schemes worldwide have recognised that certain chemicals may need to be categorised as skin or respiratory sensitisers. Although differing in detail, the schemes use similar criteria for designating materials as sensitisers, based on either direct evidence from exposed humans or the results of predictive guinea pig tests. In the case of respiratory sensitisation, however, there are currently no acceptable animal test methods. With an increasing understanding of cellular immunology in general, and of immune responses in skin and respiratory sensitisation in particular, several laboratories have recently been developing more objective, immunologically-based tests. For skin sensitisation, the two most promising methods are the murine local lymph node assay (LLNA) and the mouse ear swelling test (MEST). Both assays have undergone inter-laboratory validation and it has been shown that they are able to detect reliably moderate to strong sensitisers. The 1992 update of the OECD test guideline for skin sensitisation suggests the use of the LLNA or MEST as a first stage of testing; if a positive result is seen in either assay, a chemical may then be designated (and classified) as a potential sensitiser and it may not be necessary to conduct a guinea pig test. However, if a negative result is obtained, a guinea pig test must be performed. For respiratory sensitisation, although certain guinea pig models of asthma appear to be predictive of the known human response to sensitisers such as diisocyanates and acid anhydrides, the measurement of changes in serum IgE antibodies in mice treated topically with chemicals may represent a simpler and more accurate method of designating chemicals as respiratory sensitisers.

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

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 et al. (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.

Studies on the contact sensitizing activity of dithranol (anthralin) and 10‐butyryl dithranol (butantrone)

The contact sensitizing activity of dithranol and butantrone (10-butyryl dithranol) was studied in 3 animal models: the guinea pig maximization test (GPMT), the closed patch test (CPT), and the mouse ear swelling test (MEST) in 2 different mouse strains. In the GPMT, both dithranol and, to a greater extent, butantrone showed sensitizing potential. Because butantrone was less irritant, the concentrations used were 10x higher than those of dithranol. In the CPT, only butantrone was slightly positive. In the MEST, with both CF-1 and Balb/c mice, dithranol caused less swelling of the test ear after challenge than butantrone. According to the evaluation criteria of the MEST, only butantrone caused sensitization in 50% of the CF-1 mice and in 40% of the Balb/c mice. Thus, the GPMT was the only test which indicated the minor contact sensitizing potential of dithranol. On the other hand, the 10-butyryl analogue of dithranol showed undoubtedly stronger contact sensitizing potential than the parent compound in all tests. Therefore, as compared to dithranol, an increased risk of sensitization should be considered.

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.

Comparison of a radioisotopic incorporation method and the mouse ear swelling test (MEST) for contact sensitivity to weak sensitizers

A radioisotopic incorporation assay utilizing [ 125I]iododeoxyuridine was compared to the standard mouse ear swelling test (MEST) for the strong sensitizers dinitrofluorobenzene and oxazolone, and for the three weak sensitizers ethylenediamine (EDA), glutaraldehyde, and nickel sulfate. Mice were sensitized epicutaneously on the abdomen for 4 consecutive days prior to challenging the left ear with the test agent and the right ear with the vehicle. A comparison of the mean difference between the test and the control ears showed that measuring reactivity 48 hr postchallenge on Day 7 is the most sensitive time period in the radioisotopic incorporation method. Both the isotopic and MEST assays gave positive results with the potent sensitizers, although the response detected by isotopic labeling of emigrating cells was up to 1000-fold greater than that determined by ear swelling measurements. No response was detected to the moderate to weak sensitizer EDA in either assay. Reactivity to glutaraldehyde was not detected by the radioisotopic assay but was minimally responsive and significant by the MEST. The opposite was true for nickel sulfate where minimal but significant reactivity was seen in the isotopic assay but not in the MEST. Although the radioisotopic assay had the advantages of being more quantitative and of having improved sensitivity, it was of no greater value than the MEST for detecting weak sensitizers. It was concluded that the mouse was not a suitable model for routinely detecting reactivity to weak sensitizers regardless of which of the two assays were used.

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

Development and validation of an alternative dermal sensitization test: The mouse ear swelling test (MEST)

Traditional predictive tests for dermal sensitization in humans use the albino guinea pig as a model. A number of factors make the prospect of an alternative attractive. Guinea pig designs are labor intensive, require significant animal, caging, and husbandry resources, and are expensive. Extensive development and validation was conducted of an alternative using swelling of mouse ears as a quantitative end point. Ten strains of mice, ten age group, both sexes, induction forms (number, route, timing), the use of an adjuvant, different vehicles and intervals to challenge, two induction sites, and three measurement intervals were evaluated. A methodology was developed for preparing induction sites to increase test sensitivity. A small battery of standard compounds was used to evaluate these design variables and a final test design was developed. The basic process was also demonstrated to occur in rats and guinea pigs and to be dose responsive. The final mouse ear swelling test (MEST) design was used to evaluate 72 materials representing a broad spectrum of chemicals and testing problems. These included 49 known positives and 23 known negatives. Guinea pig maximization test data on 37 of these resulting by studies conducted in our laboratories, along with closed patch guinea pig and human test data on many of these compounds, are also reported here for the first time. The MEST correctly identified 71 of 72 materials as potential human sensitizers or nonsensitizers. Additionally, both the efficacy of an occluded patch induction method and the duration of responsiveness of mice were evaluated. In the studies, the MEST was found to be an accurate, sensitive, and efficient alternative test design for evaluating delayed-contact sensitization.