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.     

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.     

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.     

Guinea pig maximization test: Podophyllum lignans

The guinea pig maximization test was used to study the sensitizing potential of Podophyllum lignans; 0.5% of each drug in propylene glycol was used for induction and 0.01%, 0.05% 0.1% and 0.5% preparations for topical challenge. The 0.1% and 0.5% preparations caused non-allergic toxic reactions associated with the cutaneous destructive influence of these drugs; allergic responses did not occur.     

Sulphanilic acid: divergent results in the guinea pig maximization test and the local lymph node assay

The guinea pig maximization test (GPMT) has proven to be a valuable tool for the identification of the skin sensitization potential of chemicals. The method identifies a hazard which can lead in the EC to compulsory labelling of that chemical. In the present study, data on sulphanilic acid derived from the GPMT has been compared with results from a second guinea pig assay (the cumulative contact enhancement test) and the murine local lymph node assay, both of which require only topical application of chemical. Except for the GPMT, no test identified any sensitizing activity associated with exposure to sulphanilic acid. These latter results are consistent with the experience gained from substantial human exposure in an occupational setting and from which no cases of allergic contact dermatitis to sulphanilic acid have arisen over a 20-year period. In consequence, it is questioned which test protocol in practice has given the more accurate identification of sensitization hazard relevant to man.     

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.     

Sensitization studies in the guinea pig with the active ingredients of Euxyl® K 400

The preservative Euxyl K 400 consists of the 2 active ingredients, 2-phenoxyethanol and 1,2-dibromo-2,4-dicyanobutane. Sensitization studies with the guinea pig maximization test were performed with these substances, but no sensitizing capacity was demonstrated in the case of either compound.     

The sensitizing potential of various biocides in the guinea pig maximization test

Biocides are chemicals used as preservatives added to most cosmetics and toiletries and to industrial water-based products such as cutting fluids, paints and glues. There are numerous biocide formulations used in water-based metalworking fluids. All these products, characterized by a low molecular weight, can react with endogenous proteins and may induce allergic contact dermatitis. Thus, numerous reports on occupational allergic contact dermatitis have been published. The purpose of this experimental study was to compare the sensitizing potential of the various classes of biocides used in metalworking fluids by means of the maximization test of Magnusson & Kligman. The results show that, from the 9 samples tested, 7 biocides induced sensitization varying in grade from moderate to strong, while 2 benzisothiazoline-based samples did not induce allergic contact dermatitis.     

Updating the maximization test for identifying contact allergens

The maximization test for detecting contact allergens has proved to possess both sensitivity and specificity. However, modifications have become necessary because of excessive irritancy reactions to sodium lauryl sulfate. Fewer exposures are now recommended during induction and lower concentrations for challenge patch testing. Pre-testing of each subject in the panel is now standard procedure. The problem of interpreting challenge patch tests is discussed in detail.     

The effect of the guinea pig maximization protocol on the irritant response to deodorized kerosene – the excited skin syndrome *

Deodorized kerosene in a concentration of 50g/100g in pet. did not cause cutaneous inflammation in 10 control guinea pigs. The proportion of 19 guinea pigs exhibiting inflammation following exposure to deodorized kerosene in the guinea pig maximization test increased, but the increase was not significant. The response in a group of 40 animals, half exposed to tetraethyleneglycol diacrylate and half to nonylphenol polyethoxylate-6, increased (z= 3.505, p=0.004). The inflammatory response was related to concentration in both groups of animals which exhibited inflammatory responses to deodorized kerosene. The relevance of this alteration and irritant response of the interpretation of predictive to tests animals for sensitization, routine patch testing and repeated insult contact dermatitis is discussed.     

Zinc effects on nickel dermatitis in the guinea pig

Prevention of NiSO₄ induced allergic, contact dermatitis (ACD) using ZnSO_{4 in} drinking water was studied in a guinca pig model Without ZnSO₄ intervention, nickel (Ni)-exposure resulted in significantly higher (p<0.05) stimulation indices (SIs) as compared to non-exposed controls, using NiSO₄ as an allergen in the lymphocyte transformation test (LTT). Oral intake of ZnSO₄ at both 250 μg/ml double-distilled deionized water (DDD) and 500 μg/ml DDD resulted in lower SIs than those of control guinea pigs drinking only DDD: the 250 μg ZnSO₄/ml group had significantly lower Sis than (p= 0.025) than controls. There was no significant correlation between intradermal test responses and the SI values of individual guinea pigs exposed to NiSo₄ Mean Zinc (Zn) concentrations in skin and in whole blood were not statistically different between the NiSO₄ exposed control and Zn supplemented groups, nor between Ni-sensitive and non-sensitive animals within groups. The rôle of Zn homeostasis role of the Langerhans cell, effect of Zn supplementation on Ni ACD in other species, and possible blocking effects of other metals should be investigated in future studies.     

The sensitizing capacity of multifunctional acrylates in the guinea pig

The multifunctional acrylates used in ultraviolet (UV) curable resins act as cross-linkers and "diluents". They are usually based on di(meth)acrylate esters of dialcohols or tri- and tetra-acrylate esters of polyalcohols. In UV-curable coatings, the most commonly used are pentaerythritol triacrylate (PETA), trimethylolpropane triacrylate (TMPTA) and 1,6-hexanediol diacrylate (HDDA). In other uses, such as dental composite resin materials, the dimethacrylic monomers based on n-ethylene glycol are the most useful. The sensitizing capacity of various multifunctional acrylates and their cross-reactivity pattern have been investigated with the guinea pig maximization test. The tests show that BUDA (1,4-butanediol diacrylate) and HDDA are moderate to strong sensitizers and that they probably cross-react with each other. The n-ethylene glycol diacrylates and methacrylates tested are weak or non-sensitizers. Tripropylene glycol diacrylate (TPGDA) is a moderate and neopentyl glycol diacrylate (NPGDA) a strong sensitizer, whereas neopentyl glycol dimethacrylate is a non-sensitizer. The commercial PETA is a mixture of pentaerythritol tri- and tetra-acrylate (PETA-3 and PETA-4). PETA-3 is a much stronger sensitizer than PETA-4. Simultaneous reactions were seen between PETA-3, PETA-4 and TMPTA. The oligotriacrylate OTA 480 is a moderate sensitizer, but no concomitant reactions were seen with PETA-3, PETA-4 or TMPTA. Of the multifunctional acrylates tested, the di- and triacrylic compounds should be regarded as potent sensitizers. The methacrylated multifunctional acrylic compounds are weak or non-sensitizers.     

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.     

Sensitization and testing of guinea pigs with cobalt chloride

The guinea pig maximization test was used to study the sensitizing potential of cobalt chloride; 1 % CoCl2 was used for injection and 5 % for topical application. The animals were challenged twice; in series I epicutaneous testing was performed 3 weeks after sensitization and intradermal testing after yet another week. In series II the reverse schedule was used, i. e. intradermal testing after 3 weeks and epicutaneous after 4 weeks. CoCl2 was found to be a grade V allergen. The differences between cobalt-exposed and control animals (which had been treated with Freund's adjuvant, petroatum, occlusion, etc.) were statistically significant (P less than 0.001). however, the administration of cobalt at the first challenge testing caused this difference to diminish.     

Studies on contact hypersensitivity in the guinea pig The cumulative contact enhancement test

A method to determine the quantitative induction and challenge of the allergenicity of externally applied toiletories and cosmetics, including their components, is described.
The experiment used oil-soluble cinnamic aldehyde and water-soluble formalin as allergens, and guinea pigs as the experimental animals. A high sensitization method resulted, carried out as follows. A 24-h closed patch is attached to the skin every other day over a period of 2 weeks (a total of 4 applications). Freund's complete adjuvant is administered intradermally just before the 3rd application of the patch. The challenge step is performed by directly applying the test material.
This method was compared with other allergenicity evaluation methods. As a result, this method was found to be in no way inferior in sensitization performance to the other methods. The method was used cm perfume mixtures and tested for its evaluation effectiveness. It proved to be satisfactory.     

Nonspecific hypersensitivity: false-positive responses with the use of Freund's complete adjuvant

While conducting a guinea pig sensitization protocol, using the maximization test, it was discovered, at challenge, that the test animals were more responsive to the vehicle (acetone) than they were to the proprietary lest material. During rechallenge, conducted to clarify the specific immune status of the test animals, it was determined that they were also hyperreactive to an alternate vehicle (diethyl phthalate), to which they were naïve. This bizarre set of data is presented and it is suggested that this type of response is the prototype for the presence of false-positive responses experienced by loxicologists using this test. The test conditions imposed on the immune system by the maximization test that could result in these anomalous results are discussed. These data suggest that investigators need cautiously to interpret data that are produced by the injection of Freund's complete adjuvant (FCA).     

Contact urticaria from rat tail, guinea pig, streptomycin and vinyl pyridine

Four cases of contact urticaria are described due 10 rat tail, streptomycin, guinea pig and 4-vinyl-pyridine.     

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.