Sensitization studies with mometasone furoate, tixocortol pivalate, and budesonide in the guinea pig

Mometasone furoate is a new corticosteroid, synthesized to have an improved ratio of anti-inflammatory potential to adverse effects. The guinea pig maximization test was used to determine the sensitizing capacity of mometasone furoate, and also to investigate cross-reaction patterns in animals sensitized to tixocortol pivalate and budesonide, respectively. Tixocortol pivalate was shown to be a sensitizer in the guinea pig, but cross-reactions to other tested corticosteroids wore not observed. Furthermore, no sensitizing capacity could be demonstrated for budesonide or mometasone furoate.     

Dose-response studies of contact allergens using 3 guinea pig models

A multi-dose-response induction protocol for the guinea pig maximization test (GPMT), including a statistical computer program, has earlier been developed to improve the power of predictive tests for identification of contact allergens. This dose-response protocol, with 2 modifications (i.e., increased number of animals in each group and increased number of challenge concentrations) was evaluated in the GPMT, the cumulative contact enhancement test (CCET) and the Freund's complete adjuvant test (FCAT), using potassium dichromate and hydroxycitronellal as model contact allergens. Application of the dose-response protocol on the CCET and the FCAT resulted in either monotone or non-monotone curves with significant dose-response. However, application of the dose-response protocol on the GPMT gave curves with no significant dose-response. The protocol makes it possible to obtain an EC50 value, thus improving the possibility of ranking contact allergens, which is of substantial use for risk assessments. The dose-response protocol could benefit from a few adjustments: a wider span in the induction doses; change to simultaneous increase in intradermal and topical induction doses to obtain a proper dose-response for the GPMT; the addition of further challenge concentrations. In addition the computer program should allow calculation of threshold concentration for sensitization and EC50 value for a non-monotone curve.     

Experiences with Freund''s complete adjuvant test (FCAT) when screening for contact allergens in colophony

A procedure, using Freund's complete adjuvant test (FCAT), for the determination of the allergenic potential of fractions and components in colophony of the gum rosin type is described and discussed. Gum rosin was shown to be a potent sensitizer in 11 test series (153 animals). FCAT is compared with the guinea pig maximization test (GPMT). Gum rosin was a potent sensitizer according to this method as well. The FCAT method was found to be advantageous over the GPMT method in that it is technically simpler to use and a smaller amount of test substance is needed. However, closed challenge was preferred to the prescribed open challenge. The importance of statistical evaluation of the results obtained in predictive testing is stressed.     

Patch-testing with serial dilutions of tixocortol pivalate and potential cross-reactive substances

Of patch-tested patients with dermatitis, 4-5% are allergic to corticosteroids. Four groups of corticosteroids are recognized (A-D), where substances from the same group may cross-react. We investigated the potential cross-reactivity pattern and dose-response relationship for several corticosteroids from group A. We also included the corresponding aldehyde to hydrocortisone, as this degradation product has been proposed to be immunogenic. Eleven patients shown to be allergic to tixocortol pivalate were patch-tested with several corticosteroids from group A, as well as with the aldehyde, all in serial dilutions. All 11 reacted to both tixocortol pivalate and hydrocortisone. The dose-response relationship for the corticosteroids tended to be similar to sensitizers lacking anti-inflammatory potential. Patients with simultaneous reactions to many substances had high patch-test reactivity to tixocortol pivalate and hydrocortisone, while patients with few such reactions showed low reactivity (p=0.001 and 0.003, respectively). Several patients reacted to the aldehyde, supporting the theory that it is an intermediate in sensitization.     

Patch testing with corticosteroid mixes in Europe. A multicentre study of the EECDRG

This study investigated whether a corticosteroid mix containing tixocortol pivalate, budesonide, and hydrocortisone-17-butyrate could detect contact allergy to corticosteroids. 2 corticosteroid mixes, 1 with a high (mix I) and 1 with a low (mix II) concentration and the 3 individual constituents, each at 2 concentrations, were inserted into the standard series of 16 participating clinics. Tests were read on day (D) 3 or 4. 5432 patients were tested, and 110 (2.0%) had positive reactions to at least 1 of the 8 test preparations. Of the 8 preparations, mix I identified most allergic patients, followed by mix II, budesonide 0.10%, budesonide 0.002%, and tixocortol pivalate, both concentrations (1.0 and 0.10%) tracing the same number. With the mixes, 53.2-59.6% of tixocortol pivalate allergy was missed. 47 patients were allergic to either concentration of tixocortol pivalate, 25% of these only to 1.0% and another 25% only to 0.10%. Testing with mix I and tixocortol pivalate 0.10% picked up 98/110, testing with tixocortol pivalate 1.0% and 0.10% and budesonide 0.10% picked up 105/110. 3379 patients were read on both D3 or D4 as well as on D7. Without a late reading (D7), up to 30% of contact allergy to corticosteroid markers was missed.     

Sensitizing potential of acetaldehyde and formaldehyde using a modified cumulative contact enhancement test (CCET)

The contact allergenic activity of acetaldehyde was investigated with a modified cumulative contact enhancement test (CCET) method in guinea pigs. Possible cross-reactivity between acetaldehyde and formaldehyde was also studied. In contrast to the original CCET protocol, we used sham-treated controls and the chemicals were tested with closed epicutaneous application at 1st challenge. The suitability of the method was verified with formaldehyde and the results were comparable with those previously found with the guinea pig maximization test (GPMT). For the 1st time, acetaldehyde was shown to be a contact allergen in predictive tests. No cross-reactivity was observed between acetaldehyde and formaldehyde. Acetaldehyde seems to be a rare sensitizer in man. However, its allergenic activity should be considered, since it might be present as an impurity in ethoxylated surfactants. As the CCET protocol involves topical induction and challenge, we regard the modified version as well suited to evaluation of the contact allergenic potential of chemicals.     

Screening for corticosteroid contact sensitivity

3 corticosteroids have so far been tried as markers for corticosteroid contact sensitivity: hydrocortisone, tixocortol pivalate and hydrocortisone-17-butyrate. The present study compared these steroids for screening in addition to a standard patch test series. Of 727 patients, 28 (3.9%) reacted to tixocortol pivalate and 10 (1.4%) to hydrocortisone-17-butyrate; hydrocortisone gave an allergic reaction in 2 of 521 (0.4%) patients. Serial dilutions suggested that tixocortol pivalate, not marketed in Finland, caused allergic reactions which could possibly be cross-reactions to hydrocortisone. In contrast to previously published data, frequent cross-reactions occurred with hydrocortisone-17-butyrate and tixocortol pivalate. All allergic reactions to other corticosteroids found by testing with tixocortol pivalate concurred with reactions to hydrocortisone-17-butyrate. The study suggests that the most effective choice for routine testing for corticosteroid contact sensitivity would be both tixocortol pivalate and hydrocortisone-17-butyrate.     

Detection of contact hypersensitivity to corticosteroids in allergic contact dermatitis patients who do not respond to topical corticosteroids

The delayed hypersensitivity development against topical corticosteroids which are used in allergic contact dermatitis (ACD) treatment is an important clinical problem. In our study, 41 ACD patients who did not show any response to topical corticosteroid treatment were patch tested with corticosteroid series and the commercial preparations of corticosteroids and their vehicles. In corticosteroid series, there were budesonide, bethametasone-17-valerate, triamcinolone acetonide, tixocortol pivalate, alclomethasone-17-21-dipropionate, clobetasole-17-propionate, dexamethasone-21-phosphate disodium and hydrocortisone-17-butyrate. We detected positive reaction to corticosteroids in 9 of our cases (22%) (5 single and 4 multiple). The sensitivity was mostly produced by tixocortol pivalate (6 patients). This was followed by triamcinolone acetonide (2 patients) budesonide (2 patients), alclomethasone dipropionate (2 patients), dexamethasone 21 phosphate disodium (2 patients) and betamethasone-17-valerate (1 patient). As a result, it should not be forgotten that the corticosteroids used to treat ACD patients may cause ACD themselves. In ACD patients who did not respond to corticosteroid treatment, routinely applying patch test with corticosteroids should be helpful in directing the treatment.     

The value and limitations of rechallenge in the guinea pig maximization test

The guinea pig maximization test (GPMT) has played a primary rôle in the evaluation the evaluation of potential skin contact sensitizers for 25 years. In the OECD Guideline 406 from 1993, it is specifically suggested that equivocal results from the initial challenge in the GPMT should he evaluated further with a repeated challenge. However, there exist few published rechallenge data and the guideline does not describe how rechallenge data should be interpreted. In this paper, we have used examples from published results to illustrate both the positive value and the limitations of repeated challenges, including cross challenge. Testing with modified concentrations may also help to indicate whether or not the response is allergic in nature, particularly where there has been a low level of skin reaction observed in shamtreated controls, or where a low level of skin reaction is the dominant response in the test animals. In conclusion, the data presented demonstrate that, as a tool for the investigation of skin sensitizing potential, the GPMT can benefit from an experienced scientific evaluation of rechallenge data, but that this information should not be treated in a mechanistic fashion.     

Corticosteroid-induced contact dermatitis: a pragmatic approach

It is only in the past 10 years that the allergenic potential of topical corticosteroids has been fully realized. This has an important impact on the management of patients with chronic eczematous eruptions. Nonhalogenated topical steroids are more frequent sensitizers than halogenated molecules. Tixocortol pivalate and budesonide should be added to the standard series of patch test allergens. The topical steroid products that the patient has used should also be tested. If a patient has a positive reaction to tixocortol pivalate and/or budesonide then further patch testing with a commercial corticosteroid series should be undertaken.     

Reactivity of tixocortol pivalate-positive patients in intradermal and oral provocation tests

Pivalone®/tixocortol pivalate commonly yields positive reactions in the patch test series. The clinical relevance of these positive reactions was investigated in more detail. In the standard patch test series 5.6% (73 of 1306) ofthe patients were positive to corticosteroids. 5.2% to 0.1% tixocortol pivalate in ethanol (Pivalone® nasal spray diluted 1:10) and 2.3% to 1% hydrocortisone butyrate in ethanol. Some ofthe patients were tested in parallel with Pivalone® and 1% tixocortol pivalate in petrolatum. The former test reagent yielded some false-positive reactions, whereas with the latter, some allergic responses were missed. Intradermal tests with the succinate esters of hydrocortisone, methylprednisolone and prednisolone were performed with 52 patients positive to Pivalone®. Of these 76.9% (40 of 52) were positive in the intradermal tests; 38 to hydrocortisone. 35 to methylprednisolone and 30 to prednisolone. Twelve patients who had been positive in the intradermal tests were challenged orally with corticosteroids and they all showed positive reactions to hydrocortisone, methylprednisolone or prednisolone. The patients developed localized reactions at the sites of previous eczema or positive skin tests or diffuse erythema or exanthema. The oral doses of hydrocortisone eliciting positive delayed skin reactions ranged from 20 to 200 mg. Reactivity to tixocortol pivalate is closely related to sensitivity to hydrocortisone, methylprednisolone and prednisolone, but high oral doses of these corticosteroids may be required to produce allergic symptoms.     

Corticosteroid contact hypersensitivity: what vehicle and concentration?

The correct concentration and vehicle for patch Letting with corticosteroids is in many instances not known. Tine results of this study Suggest that 1% in ethanol should be the initial choice, unless it can he shown that petrolatum as a vehicle is as sensitive (tixocortol pivalate and budesonide). We could find no evidence for the anti-inflammatory effects of corticosteroids inhibiting the patch test al higher concentrations. Using ethanol as the vehicle resulted in reactions developing at earlier time points than with petrolatum.     

Patch testing to detect corticosteroid allergy: is it adequate?

Whilst patch testing with corticosteroids in ethanol is more sensitive than either petrolatum or the cream formulation, the frequency of false-negative reactions is not known. We have compared patch testing with corticosteroids at 1% in ethanol with intradermal (i.d.) tests using Img cortico-steroid suspended in normal saline. Patch tests with tixocortol pivalate and budesonide detected all patients allergic to hydrocortisone and budesonide, respectively. For other corlicosteroids, the use of ethanol as a vehicle resulted in both false-positive and false-negative reactions. In particular, patch tests with hydrocortisone-17-butyrate missed 30% of all positive reactions detected by i.d. testing. There may be a case for advising the avoidance of this steroid in all patients who are positive on patch testing to tixocortol pivalate and budesonide.     

Corticosteroids

Up to 5% of dermatitis patients are allergic to corticosteroids. Because such allergy may be difficult to suspect due to the anti-inflammatory action of the corticosteroid, markers for corticosteroid allergy should be present in any standard series. Budesonide and tixocortol pivalate are two such markers, and they seem to detect a majority of corticosteroid allergy. The patch test concentration for a given corticosteroid may be crucial. A false-negative reaction may follow despite the patient being allergic, if too high a test concentration is used, because of the anti-inflammatory action of the corticosteroid. Patch test readings must be performed not only on Day 3 or Day 4 but also on a late occasion, i.e., Day 7 after test application, also because the anti-inflammatory action may suppress an allergic reaction at an early reading. Once a patient has reacted to a corticosteroid, an extended corticosteroid series should be tested, so that information may be given on which corticosteroids to use and, above all, which corticosteroids to avoid.     

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.     

Screening for corticosteroid contact hypersensitivity

To evaluate which corticosteroids are most useful for the detection of corticosteroid contact allergy in our population, 2123 patients were patch tested with a series of 6 corticosteroids in parallel with a standard series, and other relevant investigations, 127 patients (5.98%) were allergic to one or more corticosteroids; 96 to tixocortol pivalate, 51 to hydrocortisone butyrate, 47 to budesonide, 11 to betamethasone valerate, 11 to clobetasone butyrate and 8 to clobetasol propionate, 511 patients with negative patch tests to the limited corticosteroid series were in addition tested to a further 12 corticosteroids; only 1 of these patients reacted to a corticosteroid. A combination of tixocortol pivalate and budesonide thus detected 91.3% of corticosteroid-allergic subjects. We believe that both these allergens should be included in the standard series and that there may be a case for extending this further.     

The benefit of patch testing with a corticosteroid at a low concentration.

BACKGROUND: Patch testing with corticosteroid marker molecules is advocated because testing with all available corticosteroids is impossible in clinical practice. Most commonly used are budesonide, tixocortol pivalate, and hydrocortisone-17-butyrate. We have been patch testing not only with the three markers, but also with two corticosteroid mixes, each consisting of different concentrations of the three markers. OBJECTIVE: We describe a patient allergic to tixocortol pivalate, who was diagnosed by using a lower patch test concentration that recommended, 0.1% in petrolatum, as well as a weak corticosteroid mix, 0.202%. METHODS: The patient was patch tested to a standard series, including the two corticosteroid mixes and its three constituents. RESULTS: None of the corticosteroid preparations were positive on the first ordinary reading day, day 3, whereas both tixocortol pivalate at 0.1% and the corticosteriod mix at 0.202% were positive on the second ordinary reading day, day 7, whereas all tested corticosteroids in the standard series gave positive reactions on d10. CONCLUSION: The possible benefit of patch testing with a corticosteroid at a low concentration is supported, as is the significance of late readings beyond D4.     

Stability of corticosteroid patch test preparations

This study investigated the stability of tixocortol pivalate, budesonide, and hydrocortisone-17-butyrate (Hc-17-B) when present in a mix with petrolatum and when the corticosteroids were kept separately in petrolatum. The concentrations chosen for the corticosteroids were the same as those used in a study within the European Environmental Contact Dermatitis Research Group (EECDRG), in which 2 corticosteroid mixes (1 with a high concentration and 1 with a low concentration) and the 3 individual constituents, each at 2 concentrations, were patch tested. Ethanolic solutions of each corticosteroid, as well as 2 mixtures of these 3 corticosteroids, were also made up at corresponding concentrations. The preparations were kept at room temperature, refrigerated, and deep frozen, and repeatedly for 1 year, investigations to check stability by high performance liquid chromatography were carried out. A decrease of < or =20% of the initial value at time 0 was used as the threshold for stability. The petrolatum preparations and the ethanolic solutions of budesonide and tixocortol pivalate were stable for at least the whole investigative period, irrespective of storage conditions, while Hc-17-B 1.0% in ethanol kept deep frozen was stable at least during the same period. The latter corticosteroid when kept at room temperature was stable for 3 months only.     

A new protocol and criteria for quantitative determination of sensitization potencies of chemicals by guinea pig maximization test

This paper presents precise sensitization test data of 15 chemicals with a wide spectrum of sensitization potencies, and proposes a new protocol and criteria for quantitative evaluation of sensitization potencies of chemicals. The tests were performed according to the design of Magnus-son and Kligman, changing the application concentrations for induction as well as for challenge phases. 3-dimensional relationships between mean response (or sensitization rate), induction and challenge concentrations were found in all chemicals tested. The following 2 values are proposed as a quantitative measure of sensitization potency: (a) the minimum induction concentration that induces a positive response; (b) the challenge concentration that induces a mean response approximately equal to 1.0 among the animals applied with the highest concentration for induction. Both values coincided with each other within the range of 1 order of magnitude in every compound except 2. The values varied by 5 orders or more of magnitude among the compounds, showing a wide variation of sensitization potencies among chemicals. A good correlation was found for every chemical between the value of sensitization potency thus obtained and the residual levels in causative products in human cases of allergic contact dermatitis. A new experimental protocol for obtaining values (a) and (b) is proposed.     

Contact allergy to colour developing agents in the guinea pig

Colour developing agents, derivatives of p-phenylenediamine, can cause contact allergy. Patch test reactions to more than one colour developer are sometimes seen in patients. To study whether this is due to simultaneous sensitization or cross-reactivity, guinea pig maximization tests (GPMT) with CD-2, CD-3 and CD-4 were carried out. 5 experiments were performed, using pet. or water as vehicles. When pet. was used, the challenge concentrations could be raised and cross-reactivity between the colour developers, but not with p-phenylenediamine-dihydrochloride, was revealed. When water was used as vehicle, the challenge concentrations were limited because of staining of the test sites and irritation. CD-2, CD-3 and CD-4 were found to be extreme sensitizers according to the classification by Magnusson and Kligman. The importance of using an appropriate vehicle to obtain optimal conditions for the GPMT is stressed. To study the purity and stability of the chemicals used, analysis by HPLC of the test substances at different stages of the GPMT procedure was performed. Aqueous solutions of the colour developers were found to be unstable, while pet. mixtures were stable.