Predictive evaluation in animals of the contact allergenic potential of medically important substances I. Comparison of different methods of inducing and measuring cutaneous sensitization

Groups of guinea pigs were sensitized with a 0.1% solution of dinitrochlorobenzene (DNCB) by the Draize intracutaneous method, The course of the induction process, the influence of the vehicles used and the extent to which the reactions are amenable to assessment according to objective criteria were examined. The sensitivity of the standardized Draize test was then compared with that of various other sensitization techniques, including:
The intracutaneous test with adjuvant (optimization test)
The maximization test according to Magnusson & Kligman (1969)
The epidermal sensitization test
The epidermal sensitization lest wish prior irritation of the contact site (by croton oil or sodium lauryl sulphate).
Comparison of these methods revealed that either the additional application of adjuvant or prior irritation of the contact site augmented the degree of sensitization to DNCR just as greatly as the simultaneous use of adjuvant and prior irritation of the skin, (maximization test.). The improved sensitization methods, and in particular the standardised optimization test, may prove to be of particular value for the study of so-called weak allergens.     

Identification of contact sensitizers by animal assay

Guinea pig testing constitutes the first step in evaluating the allergenicity of new chemicals and products. Some of the most commonly used animal predictive tests are reviewed. The guinea pig maximization test, which is the recommended test method in Sweden, is described in detail and the interpretation of results obtained with this test is discussed. In the guinea pig maximization test the sensitization capacity of a substance is examined by the use of maximized conditions for i he exposure, i.e. the potential ability of the material to induce a contact allergy is determined. The extent to which an allergen causes contact dermatitis in exposed persons depends on the mode of use and various environmental factors.     

How sensitizing is chlorocresol?

Chlorocresol is a biocide with widespread use in industry and pharmaceutical products. It is an occasional human contact sensitizer. The sensitizing potential of chlorocresol was judged strong using the guinea pig maximization test (GPMT) and doubtful in the less sensitive open epicutaneous test (OET). When different induction concentrations were used, the results indicated an optimal sensitizing concentration above which no further increase in the sensitization rate occurred. Rechallenge 2 weeks later showed a marked decrease in sensitivity. Consecutive human patch tests with chlorocresol 2% in pet. showed 11 reactions among 1462 patients tested, but none were explainable and reproducible during re-tests and provocative use tests, indicating that the GPMT overestimated the sensitization potential. The results from guinea pig allergy tests cannot stand alone but have to be validated by other sources of information.     

Nickel-sulphate-induced contact dermatitis in the guinea pig maximization test: a dose-response study

Nickel sulphate is a sensitizer in guinea pigs, but the frequency of sensitization varies from study to study. The dose-response relationship for NiSO4.6H2O was evaluated in the guinea pig maximization test in this study. 6 intradermal (0.01%-3.0% aq.) and 6 topical (0.25%-10.0% pet.) concentrations were chosen for induction and NiSO4.6H2O 1% pet. was used for challenge, based on the absence of skin irritation in a pilot study. Blind reading was performed. A logistic dose-response model was applied to the challenge results. At 48 h, a linear relationship was obtained between the intradermal induction dose (but not topical dose) and the response, resulting in a maximum sensitization rate of 40% after 3% i.d. The reactivity disappeared at re-challenge 1 week later. Following a booster closed patch on day 35, using NiSO4 10% pet., the animals were challenged with NiSO4 2% pet. and statistical analyses of 72-h readings revealed a non-linear dose-response relationship, giving a maximum response frequency of 40% after initial induction with NiSO4 3% i.d. and 2% topical.     

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.     

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.     

Predictive evaluation in animals of the contact allergenic potential of medically important substances

Results of the optimization method and of other methods used to assess contact allergy in laboratory animals were compared with known epidemiological data on the occurrence of hypersensitivity reactions in man. Tests were performed with preservatives {formalin, ethylenediamine and sorbic acid), drugs (penicillin G, benzocaine and sulphathiazole) and other contactants belonging to widely different chemical classes (p-phenylenediamine, triclosan, pyrazole derivatives, nickel and chrome salts, eugenol, isoeugenol and mercaptobenzothiazole). The degree of sensitization achieved in guinea pigs by the optimized procedure (intradermal test with adjuvant combination) and the maximization procedure was invariably superior to that produced by the epidermal method using prior irritation of the site of application. Both the optimized procedure and the maximization test seem to be capable of identifying contact allergens that cause hypersensitivity reactions in as few as t in 10,000 of the human population as a whole. The optimization test merits consideration as a standardized and efficiently predictive procedure.     

Primary irritant effect of 3,4,5-trichloropyridazine

A primary irritant dermatitis to 3,4,5-trichloropyridazine was observed on the hands of a chemist after accidental exposure. Healing was rapid in comparison to a report on 3,4,6-trichloropyridazine toxicity. Prompt treatment with a potent topical corticosteroid, wet soaks, and an oral antihistamine brought resolution within 14 days.     

Contact photoallergy testing of sunscreens in guinea pigs

The potential of 3 sunscreens (p-aminobenzoic acid, 4-isopropyldibenzoylmethane and homosalate) and 2 known human photoallergens (musk ambrette and tetrachlorosalicylanilide) to cause photoallergy, phototoxicity, and/or contact sensitization was determined using a guinea pig photoallergy model, as previously described by Harber and associates. The model was slightly modified by employing 6 exposures over 2 weeks and using Hill Top Chambers for application of the test material. Contact photoallergy was detected in guinea pigs treated with musk ambrette or tetrachlorosalicylanilide (TCSA), although with TCSA, a lower incidence of contact sensitivity and phototoxicity was also detected. The results of studies conducted with sunscreens showed that p-aminobenzoic acid was photoallergenic, whereas homosalate and 4-isopropyl-dibenzoylmethane (Eusolex 8020) were not. However, contact sensitization, and to a lesser degree primary irritation, was detected with Eusolex 8020 at the concentrations employed in this study. The results of these studies suggest that this guinea pig model is a suitable model for assessing the photoallergic potential of various compounds, including the sunscreens tested in this study.     

Sensitizing potential of 2-hydroxyethylmethacrylate

Guinea pigs exhibited none or slight responses to sensitization with low concentrations of 2-hydroxyethylmethacrylate in the guinea pig maximization test, while 60-100% reacted to high concentrations regardless of the vehicle used for induction. Petrolatum, water, soybean oil and a mixture of oil and 2-butanone (sbomek) were used as vehicles for elicitation. The neat methacrylate was less effective than dilutions in any vehicle, petrolatum being the best. The major determinant of the frequency of response was the concentration used for intradermal induction. An increase in frequency and in duration of responsiveness after treatment with cyclophosphamide 3 days before challenge suggests that hydroxyethylmethacrylate preferentially stimulates the suppressor cell function.     

Identification of contact allergens in C.I. Solvent Red 23 (commercial Sudan III) by chemical analysis and animal testing

C.I. Solvent Red 23, commercial Sudan III, is widely used in cosmetic products. Chemical analyses and guinea pig sensitization tests were carried out to identify its contact allergens. In the Magnusson & Kligman guinea pig maximization test, C.I. Solvent Red 23 showed 20% positive reactions. By conducting chemical analyses with HPLC and GLC, 2-naphthol (82 ppm), azobenzene (48 ppm), Sudan I (570 ppm) and many unknown impurities, as well as the main constituent pigment Sudan III (87%), were found. The chemical structure of one unknown impurity was identified as an isomer of Sudan III. We found that purified Sudan III showed no positive reaction, while the isomer elicited 30% positive reactions, in the same guinea pig test. Furthermore, cross-sensitization with p-phenylenediamine was investigated using the guinea pig test. Animals sensitized with p-phenylenediamine also showed positive elicitation reactions with purified Sudan III. From these results, the contact allergenicity of C.I. Solvent Red 23 is considered to be due to impurities, including the isomer of Sudan III, 1-(o-phenylazophenylazo)-2-naphthol. Positive reactions to Sudan III previously demonstrated in hairdressers are due to cross-sensitivity with p-phenylenediamine.     

Induction of formaldehyde contact sensitivity: dose response relationship in the guinea pig maximization test

The sensitizing potential of aqueous formaldehyde was evaluated with the guinea pig maximization test (GPMT) in two laboratories (Copenhagen and Stockholm) using different guinea pig strains. Six intradermal (0.01%-3%), and 6 topical (0.5%-20%) concentrations were used for induction, and formaldehyde 1% and 0.1% was used for challenge. The incidence of contact sensitivity depended on the intradermal, but not on the topical induction dose. Statistical analyses showed a non-monotonous (non-linear) dose response relationship. The estimated maximal sensitization rate in Copenhagen was 80% after intradermal induction with 0.65% formaldehyde; in Stockholm it was 84% after induction with 0.34%. The data from the two laboratories could be described by parallel displaced dose response curves suggesting that the guinea pig strain used in Stockholm was significantly more susceptible to formaldehyde than the strain used in Copenhagen. The EC50 (formaldehyde concentration at which 50% of the guinea pigs were sensitized) at the 72 h scoring and a 1% challenge concentration, was 0.061% in Copenhagen and 0.024% in Stockholm.     

Studies of new short-period method for delayed contact hypersensitivity assay in the guinea pig

The enhancement effect of cyclophosphamide on the delayed contact hypersensitivity reaction of chemical compounds was studied in Hartley albino guinea pigs. A series of assay procedures. combining the AP2 test (adjuvant and 24-h occlusive patch 2× test, as previously reported) with intraperitoneal cyclophosphamide administration, were examined. The newly developed method was as follows; cyclophosphamide 200 mg/kg intraperitoneal administration 3 days before the 1st sensitization of the AP2 test (cyclophosphamide. adjuvant and 24-h occlusive patch 2× test: CAP2 test). Comparing the CAP2 test with the AP2 test, the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT), the CAP2 test equally and/or better enabled the detection of allergenicities not only of strong allergens such as bromostyrol, citronellal, p -phenylendediamine and formaldehyde, but also of weak allergens such as benzyl salicylate and p -aminobenzoic acid ethyl ester. Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dennis at the skin reaction site were histopathologically observed. Cyclophosphamide effectively enhanced the delayed contact hypersensitivity reaction of weak allergens.     

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.     

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.     

A modified technique of guinea pig testing to identify delayed hypersensitivity allergens

A modified guinea pig testing technique was developed For the detection of weak allergens and allergenicity of materials unsuitable for testing by intradermal injection. This test involved the use of Freund's complete adjuvant to stimulate the immune system of the animal, and external application instead of intradermal injection of the test compound in the induction stage. The allergenicity of Sudan III, Brilliant Lake Red R and Sudan I was tested by this procedure.
In the dose-effect study of Sudan I, the dose dependency of a positive reaction of the induction and challenge concentrations was recognised.
The test was compared with three other guinea pig sensitization tests. The results obtained with this test correlated well with those obtained with the guinea pig maximization test.     

Sensitizing potential of chlorothalonil in the guinea pig and the mouse

The fungicide chlorothalonil is used extensively under several tradenames for the protection of various horticultural and fruit crops and bananas against fungal infections. It is also used as fungicide in wood preservation and as a preservative in paints. Clinical experience has shown chlorothalonil to be a contact allergen and several cases of allergic contact dermatitis attributed to chlorothalonil have been described. 2 previous guinea pig maximization test studies have shown the sensitizing potential of chlorothalonil to be high. The sensitizing property of chlorothalonil was studied by us with the predictive test methods the local lymph node assay and the cumulative contact enhancement test. In the local lymph node assay, chlorothalonil induced a dose-dependent increase in proliferation with a maximal stimulation index of 19.2 and 27.2. In the cumulative contact enhancement test, a statistically significant dose-dependent high sensitization rate was seen with a maximal sensitization rate of 100%. In conclusion, it is evident that chlorothalonil is an extremely potent contact allergen, inducing sensitization using only topical exposure on intact skin.     

Phosgene (chlorophenyl)hydrazones, strong sensitizers found in yellow sweaters bleached with sodium hypochlorite, defined as causative allergens for contact dermatitis by an experimental screening method in animals

12 young men developed allergic contact dermatitis from wearing yellow cotton sweaters. We attempted to identify the causative agents by an experimental screening method in animals. Guinea pigs were sensitized with an acetone extract of the sweater material, by means of the guinea pig maximization test (GPMT). Active ingredients were then separated from the extract, by step-by-step patch test screening of chromatographic fractions in the guinea pigs, and finally analyzed by gas chromatography-mass spectrometry (GC-MS). Although there were 2 allergens with important activity (1 in the fraction eluted from the silica gel column with hexane, and 1 in the methanol fraction), the present study is focussed on the fat-soluble allergens in the hexane fraction. GC-MS analysis revealed that 4 kinds of phosgene (chlorophenyl)hydrazones (PCPHs) were present in the hexane fraction. PCPHs prepared in our laboratory showed strong eliciting activities, not only in the guinea pigs sensitized with the extract, but also in a male volunteer sensitized by exposure to a yellow sweater during irritancy testing. Phosgene (2,5-dichlorophenyl)hydrazone, which was the main component among the PCPHs found in the sweater, sensitized guinea pigs even at the 1 ppm level. From these results, we conclude that PCPHs were one of the allergens responsible for the cases.     

Studies of new short-period method for delayed contact hypersensitivity assay in the guinea pig

A new method for delayed contact hypersensitivity assay of chemical compounds in guinea pigs, a short-period method (14 days) with a high detection sensitivity, has been developed. The new method was as follows; a combination of a Freund's complete adjuvant (FCA, undiluted) intradermal injection and a 24–h occusive patch on a guinea pig was performed 2x at an interval of 4 days and challenged by non-occlusive topical application II days after the first sensitization (with benzyl alcohol during test development). Acanthosis and spongiosis in the epidermis and mononuclear cell infiltration into the dermis were observed histopathologically at the skin reaction site. This newly developed method (adjuvant and 24–h occusive patch 2 test: AP2 test) could equally and/or better detect the allergenicities of 6 other chemical compounds (bromostyrol, citronellal, benzyl salicyfate. p -aminobenzoic acid ethyl ester, phenylenediamine and formaldehyde) as compared with the cumulative contact enhancement test (CCET) and the guinea pig maximization test (GPMT).     

Tixocortol pivalate contact allergy in the GPMT: frequency and cross-reactivity

In spite of their intrinsic anti-inflammatory properties, corticosteroids can induce contact allergy. When studying the allergenic properties of corticosteroids it has to be considered that both the allergenic and anti-inflammatory effect may influence the induction phase as well as the elicitation phase and that such effects may be dose-dependent. A multiple dose guinea pig maximization test (GPMT) was therefore used to study the dose-response relationship of tixocortol pivalate. The GPMT was conducted according to OECD guideline #406, using a multiple-dose design and test results were analysed with logistic regression analysis. There was a significant tixocortol pivalate sensitization of the test animals compared to the control group (p<0.05), after both challenge and re-challenge. The challenge with 1% tixocortol pivalate gave more positive reactions than the challenge with 3%. The highest frequency of positive animals was observed when the animals were treated with low to intermediate induction concentrations and intermediate to high challenge concentrations with tixocortol pivalate in the TRUE Test. Cross-reactivity was found between tixocortol pivalate and hydrocortisone, which was expected from their close molecular resemblance, whereas no cross-reactivity was seen between tixocortol pivalate and the 3 other corticosteroids: amcinonide, budesonide, and hydrocortisone-17-butyrate.