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.     

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.     

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.     

Sensitizing potential in mice, guinea pig and man of the preservative Euxyl K 400 and its ingredient methyldibromo glutaronitrile

The allergenicity of the preservative Euxyl K 400 and its principal allergen methyldibromo glutaronitrile (MDBGN) (1,2-dibromo-2,4-dicyanobutane) was investigated using 3 animal models; in mice, the local lymph node assay (LLNA) and in guinea pigs, the guinea pig maximization test (GPMT) and the cumulative contact enhancement test (CCET) with a dose-response protocol included. Previous attempts to define the sensitization capacity of these chemicals have given conflicting results. For comparison, the frequency and causes of positive patch test reactions to Euxyl K 400 and MDBGN were studied in patients referred to an occupational dermatology clinic. This investigation showed that Euxyl K 400 and MDBGN can give rise to contact allergy in man and that the relevant cases found mainly had similar exposure as non-occupational cases. A contact allergenic potential could be detected for MDBGN in 2 animal models, i.e., the CCET and the LLNA, and also for Euxyl K 400 in the LLNA. However, statistical analysis of the results from the GPMT with MDBGN failed to detect the sensitizing potential of this particular allergen. The results indicate that to be able to detect the allergenic potential of Euxyl K 400 and MDBGN, a predictive test method with multiple topical applications at induction is required. It is therefore important that an investigator is aware of the possibility of using various predictive test models for investigation of potential contact allergens.     

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.     

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.     

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.     

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.     

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.     

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).     

An interlaboratory evaluation of the Buehler test for the identification and classification of skin sensitizers

The correct identification of potential skin sensitizers is an essential first step in enabling a proper risk assessment to be made and to permit the implementation of appropriate risk management practices designed to avoid the induction of sensitization, Consequently, regulatory guidelines around the world demand that new substances are evaluated to assess their skin sensitization potential. There are two guinea pig test methods which are generally recognised, the guinea pig maximisation test (GPMT) and the occluded patch test described by Buehler. In different countries, one procedure seems to be more prevalent and acceptable to regulatory authorities than the other. Notably, in the European Union, the latest revision of the Annex V (Directive 92/32/EC) Test Method for skin sensitization asks that justification should be given in the situation where the notifi this paper, the validity of the Buehler protocol in the context of European legislation is critically examined. Results from two laboratories are collated. showing that the method can identify significant contact allergens, particularly those which would he registered formally as such according to European legislation. It is demonstrated that minor methodological variations can he tolerated without compromising tesi sensitivity, hut it is recommended that suitable positive control testing is the best way to ensure proper test conduct.     

Comparison of the sensitivities of the Buehler test and the guinea pig maximization test for predictive testing of contact allergy

International test guidelines, such as the Organisation for Economic Cooperation and Development (OECD) guideline #406, recommend 2 guinea pig methods for testing of the contact allergenic potential of chemicals: the Guinea Pig Maximization Test (GPMT) and the Buehler test. Previous comparisons between the methods suggested that the Buehler test was less sensitive than the GPMT although modified Buehler test protocols were used. Parallel GPMT and Buehler tests were conducted according to OECD guideline #406 using a multiple-dose design and test results were analysed using a standard logistic dose-response model. To compare the sensitivity of the 2 test procedures the test conditions were kept identical and the following chemicals with a range of sensitization potentials were tested: chloraniline, chlorhexidine, eugenol, formaldehyde, mercaptobenzothiazole and neomycin sulphate. Formaldehyde and neomycin sulphate were strong sensitizers in both tests. Mercaptobenzothiazole, eugenol and chloraniline were all strong sensitizers in the GPMT, eugenol and mercaptobenzothiazole were negative in the Buehler test and equivocal results were obtained with chloraniline. Chlorhexidine was negative in the GPMT and equivocal responses were obtained with the Buehler test. Higher induction concentrations were needed to show allergenicity in the Buehler test and for some allergens the Buehler test protocol was not sensitive enough to demonstrate allergenic potential.     

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.     

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.     

Method for assessment of experimental allergy in guinea pigs adapted to cosmetic ingredients

2 protocols are presented to predict and assess allergic reactions after application of chemicals or cosmetic products, either raw materials or finished products. The first is an open epidermal induction and challenge, the intensity of the reaction being maximized by injecting Freund's complete adjuvant into the foot pad. The second allows detection of weaker allergens and is a quasi-intradermal induction: both the adjuvant and test substance are injected into the foot pad. In both protocols, the challenge consisted of a single topical application in the lumbar region of 10 microliter of test substance and components, allowing study of cross-sensitization, the large surface available permitting 6 different contacts.     

Evaluation of contact allergy to chemicals using laser Doppler flowmetry (LDF) technique

Skin blood flow in allergic contact reactions and cross-sensitivity were evaluated using laser Doppler flowmetry (LDF) to study the dose-response relationships in phases of induction and challenge in guinea pigs. Guinea pigs were sensitized with different doses of 1-chloro-2,4-dinitrobenzene (DNCB) and challenged with different doses of DNCB and 2,4-dinitrobenzene sulfonic sodium salt (DNBS). The skin reactions were evaluated by LDF and visual reading score. The results indicated that there were dose-response relationships between the doses of DNCB and LDF measurements in both phases of induction and challenge, that there was a cross-reaction between DNCB and DNBS, and that the reactions at 24 h were greater than that at 48 h after removal of the patches. LDF may discriminate between positive patch test reactions and negative or doubtful reactions, but not between weak positive and strong positive reactions. This is because vascular dilatation and increase of flow already reaches a maximum in weak reactions. The more advanced phases are dominated by oedema formation. This is simply the nature of the inflammatory reaction, rather than a methodological error. The important point is that LDF can separate positive reactions from negative/uncertain reactions. The results indicated that LDF, as a non-invasive technique, may objectively and quantitatively evaluate the dose-response relationships of contact sensitivity of sensitizers.     

A critical commentary and updating of the guinea pig maximization test

The guinea pig maximization test (GPMT) of Magnusson and Kligman was published in 1969. Since then, a vast body of practical experience with the test has been accumulated. New information requires that certain aspects of the procedure be reevaluated, especially with regard to the interpretation of challenge results. In particular, awareness of the phenomenon of hyperirritable skin (the 'angry back' phenomenon) suggests that presently used controls are not always adequate and may overstate allergenicity owing to false-positive reactions. The control group should be exposed to a chemical insult at induction which provokes an inflammatory reaction comparable to the test substance. We present strategies to distinguish irritant from allergic responses. Allergic reactions should persist on rechallenge weeks later, while nonspecific irritant reactions generally fade and are irreproducible in particular animals. Finally, when a chemical is identified as a contact sensitizer of risk is necessary to estimate the relevance of the test result to usage in the real world.     

Hydrogenation reduces the allergenicity of colophony (rosin)

Abietic acid is the main component of rosin. It is readily oxidized by air and its oxidation products are considered to be mainly responsible for the allergenic effect. Hydrogenation of the conjugated double bonds of abietic acid decreases its susceptibility to air oxidation and would thus reduce the allergenicity of rosin. Portuguese gum rosin was therefore hydrogenated and its allergenicity was compared with that of unmodified rosin in animal experiments and by patch testing in humans. Its sensitizing potential was determined in 2 studies. No response was found according to the FCAT method while the GPMT gave significant response in the animals challenged with the highest test concentration. Hydrogenated rosin showed no eliciting activity in animals induced with unmodified rosin according to the GPMT method. A marked decrease was found in the frequency of allergic reactions to hydrogenated rosin compared to the reactions to unmodified rosin in patients with known allergy to gum rosin. We conclude that elimination of the unsaturated non-aromatic compounds by hydrogenation considerably reduces the allergenicity of Portuguese gum rosin.     

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.