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.     

Sensitizing potential of 14 mono (meth) acrylates in the guinea pig *

The aim of this study was to investigate differences in the sensitizing potential of 14 mono(meth)acrylates, when tested by the guinea pig maximization test (GPMT) and Freund's complete adjuvant test (FCAT) with an identical, intradermal induction concentration. A new grading classification of the sensitization potential is proposed. Mono(meth)acrylates show a wide range of sensitizing potential. Compared with other (meth)acrylates, methyl methacrylate is a moderate sensitizer. Attention was paid to concomitant sensitization of additives. In 9 of 16 FCATs, concomitant sensitization occurred to the inhibitors hydroquinone and p-methoxy phenol.     

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.     

Air oxidation of d-limonene (the citrus solvent) creates potent allergens

Products containing as much as 95% of d-limonene are used for, e.g., degreasing metal before industrial painting and for cleaning assemblies. Experimental studies on the sensitizing potential of limonene show diverging results. In a previous study, we found that the sensitizing potential of d-limonene increased with prolonged air exposure. The aim of this study was to make further chemical analyses, to identify compounds formed by air exposure of d-limonene and to study their allergenic potential. d-limonene was found to be a sensitizer after prolonged exposure to air according to 2 Freund's complete adjuvant test (FCAT) experiments and 1 guinea pig maximization test (GPMT) study. No significant response was obtained to d-limonene not air exposed, even if the animals were sensitized to oxidized d-limonene. 5 main oxidation products of d-limonene were identified. (R)-(-)-carvone and a mixture of cis and trans isomers of (+)-limonene oxide were found to be potent sensitizers, while no significant reactions were obtained in the animals induced with a mixture of cis and trans isomers of (-)-carveol. It can be concluded that air oxidation of d-limonene is essential for its sensitizing potential, and that potent allergens are created.     

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.     

Isolated colophony allergens as screening substances for contact allergy

Rosin has a complex chemical composition. The aim of this study was to investigate if it is possible to define 1 or 2 compounds as the rosin allergens. 2 compounds, 13, 14(β)-epoxyabietic acid and 7-oxodehydroabietic acid, identified in gum rosin and tall oil rosin, were used as screening substances for patch testing in addition to the standard series. The rosin patch detected more cases of contact allergy than the isolated allergens did. Our suggestion is to patch test with a preparation of gum rosin, as well-defined as possible, for screening. The content of oxidation products should be kept at a constant and rather high level, since these are the main allergens. A rosin series for additional testing of patients with allergic contact dermatitis suspected to be caused by rosin could he a valuable tool. Gum rosin and tall oil rosin from different countries should be included, since the amounts of allergens in rosin varies due to source and mode of production. Testing with identified allergens from modified rosin products and with modified rosins could also be performed.     

Maleopimaric acid – a potent sensitizer in modified rosin

The allergenicity of the maleic-modified rosins and their esters has been studied. The unesterified resins are mainly used in paper size and the esters in printing inks, varnishes and adhesives. The levopimaric-maleic anhydride Diels-Alder adduct (maleopimaric acid) is the main component obtained in the maleic-modified rosins. This compound was synthesized and its structure was determined. Its sensitizing potential was investigated in guinea pigs according to different methods. It was shown that maleopimaric acid is a very potent sensitizer, comparable with the strongest allergen isolated from unmodified gum rosin. The allergen may also be present after esterification unless the process is carried out to completion. The animals sensitized to maleopimaric acid did not react to unmodified rosin, which shows that maleopimaric acid is structurally different from the allergens in rosin.     

Cross-reactivity to palladium and nickel studied in the guinea pig.

In recent years there have been several reports on concomitant patch test reactions to palladium and nickel, which belong to the same group in the periodic table. Exposure to palladium mainly takes place via dental alloys and jewelry. However, the clinical relevance of simultaneous reactivity to these metals is unknown. To elucidate the question of cross-reactivity, guinea pigs were induced with palladium or nickel and simultaneously challenged with palladium and nickel. Animals sensitized to palladium according to the guinea pig maximization test method (GPMT) or to a new method by van Hoogstraten & Scheper (H&S) reacted to palladium as well as to nickel. On the other hand, animals sensitized to nickel according to H&S reacted to nickel but not to palladium. The GPMT shows that palladium is a more potent sensitizer than nickel: could palladium be the primary sensitizer in humans?     

Contact allergy to dehydroabietic acid derivatives isolated from Portuguese colophony

7-oxodehydroabietic acid and 15-hydroxydehydroabietic acid were isolated as their methyl esters from Portuguese colophony of the gum rosin type and identified as contact allergens. Another oxidation product of dehydroabietic acid, 15-hydroxy-7-oxodehydroabietic acid, was synthesized and identified as a component of Portuguese gum rosin. 7-oxodehydroabietic acid was found to a be a grade III allergen according to the GPMT method. Guinea pigs induced with gum rosin showed only a low response to the isolated compounds, while patients with a known allergy to gum rosin reacted to a greater extent. The results imply that the content of oxidized dehydroabietic acids in gum rosin is too low to give a marked sensitization in the animals. However, the patients might have come in contact with the allergens in technically modified rosins. The compounds showed a pattern of cross-reactivity in the animal experiments as well as among the patch tested patients.     

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.     

Guinea pig maximization test of the bark extract from pawpaw, Asimina triloba (Annonaceae)

Several acetogenins, characteristic of the Annonaceae, have been found to exhibit pesticidal and feeding-deterrent activities against a variety of pests and are undergoing commercial development as pesticides. The sensitizing potential of one of these plants, pawpaw. Asmina triloba Dunal, was examined in the current study. The potential of Fraction F020 (a pesticidal crude extract of pawpaw stem bark) to sensitize and elicit an allergic contact dermatitis response was determined by using a modified guinea pig maximization test (GPMT). Fraction F020 is a weak sensitizer and the active compound, asimicin, a trihydroxy-bistetrahydrofuran fatly acid γ-lactone, is a weak irritant.     

Sensitizing potential of 12di(meth)acrylates in the guinea pig *

The aim of this study was to investigate the sensitizing potential of 12di(meth)acrylates, when tested in the Guinea Pig Maximization Test (GPMT) and Freund's Complete Adjuvant Test (FCAT). In these tests the same (molar) concentration was used for intradermal induction. The results (see table, below) show that dimethacrylates are moderate to strong sensitizers in the guinea pig. In the group of diacrylates, no sensitization to diethylene glycol diacrylate was observed. 1,2-ethanediol diacrylate and 1,6-hexanediol diacrylate sensitized a number of animals. The challenge reaction pattern of the FCAT with 1,4-butanediol diacrylate, neopentane diol diacrylate and 1,5-pentanediol diacrylate differed from that commonly observed with sensitizers. Positive reactions were seen only to the maximum non-irritant concentration at 24 h in the challenge at day 21. At 48 h and in the second challenge at day 35, the reactivity had decreased or disappeared. This different reaction pattern makes it difficult to classify these monomers with certainty as sensitizers. In most of the FCATs, a decrease in reactivity in successive challenges was found. Possible explanations for this decrease and for the different challenge reaction patterns of some diacrylates are discussed. The skin irritant capacity of di(meth)acrylates was estimated after one open application. Diacrylates are very strong irritants on guinea pig skin. Dimethacrylates are weak irritants.     

Methyl esterification of 15-hydroperoxyabietic acid does not affect the patch-test result in colophonium allergic patients

Colophonium (gum rosin) consists of numerous compounds. We have previously shown that abietic acid (Fig. 1), the major compound in gum rosin, is oxidized to strong contact allergens at air exposure (1). The most potent allergen identified is 15-hydroperoxyabietic acid (15-HPA, Fig. 1), which was isolated as its methyl ester (Fig. 1) and used for patch testing (2, 3). The aim of this study was to confirm the previous postulate that methyl esterification of the carboxyl group in 15-HPA does not affect the allergenic activity.     

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.     

Occupational dermatoses at a film laboratory

During the period January 1980-June 1981, an investigation was made of skin diseases among employees of a film laboratory. The investigation was carried out in 5 stages. (I) Questionnaire (II) Clinical examination and interview with all employees who stated that they had had skin disease at some time (III) Patch testing at the laboratory of all cases of suspected occupational dermatoses, and control tests in hospital of 200 non-exposed eczema patients (IV) Investigation of protective gloves by patch testing and in a chemical bath (V) Allergy test on guinea pigs (GPMT) of one agent (PBA-1). The firm had 114 employees and the questionnaire was answered by 103. Altogether 43 persons at some time had been exposed to chemicals in their work, and 21 of them had had occupational dermatoses. In 12 cases, contact allergy was found to one or more of the photographic chemicals CD-2, CD-3, Metol and PBA-1. GPMT with PBA-1 showed it to be a potent sensitizer. The colour developing agents may also give rise to lichenoid reactions and 2 such cases were seen in this investigation. Other causes of occupational dermatoses are indicated, as well as the occurrence of non-occupational skin diseases.     

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.     

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.     

Sensitization capacity of ethylenediamine in the guinea pig and induction of unresponsiveness

The sensitization capacity of ethylenediamine was examined by the guinea pig maximization test and compared with dinitrochlorobenzene. Ethylenediamine was found to be a potent sensitizer. An attempt at oral induction of unresponsiveness (tolerance) was unsuccessful with ethylenediamine and DNCB and the same negative result was obtained with intravenous injection of ethylenediamine. Only by the intravenous injection of dinitrobenzene sulfonic acid was unresponsiveness successfully established in 90% of the guinea pigs.     

Patch testing with allergens from modified rosin (colophony) discloses additional cases of contact allergy

Most rosin (colophony) used today is chemically modified. Will contact allergy 10 modified rosin be overlooked when testing is performed with only unmodified rosin in the standard series? 2 components from modified rosins. maleopimaric acid (MPA) and glyceryl 1-monoahietale (GMA), earlier identified us contact allergens, were added to the patch lest standard series. Some commonly-used modified rosin products were also tested. Positive reactions were observed to the modified components MPA and GMA. Of the patients with positive reactions to MPA and GMA. several did not react to unmodified rosin in the standard series. No reaction was observed to the modified rosin products without a concomitant reaction to unmodified rosin. The positive reactions observed to the modified rosins may be due to residual unmodified material in the product, but a possible contribution from allergenic modified rosin components cannot be disregarded. When u patient's history indicates a rosin allergy, but a negative response to unmodified rosin in the standard series is observed, additional testing with GMA and MPA or, second best, with glycerol-esterified rosin and maleic-anhydride-modified rosin is recommended. Some modified rosin products, which could be used for additional testing, are suggested.     

Use of the local lymph node assay in the evaluation of the sensitizing potential of pharmaceutical process intermediates

The murine local lymph node assay (LLNA) has recently been developed to determine the contact sensitization potential of chemicals. Since its original development, the LLNA results have been the subject of extensive comparisons with guinea pig and human data. The investigations described here were designed to explore the ability of the LLNA to identify accurately, pharmaceutical process intermediates (PIs) known to cause contact allergy in humans. To that end, 16 PIs previously tested in the guinea-pig maximization test (GPMT) were tested in the LLNA. Another PI known to be a contact sensitizer in humans was tested only in the LLNA. Cases of contact sensitization in humans were reported only for PIs that were extreme sensitizers in the GPMT and had low EC3 values (concentration of the test substance required to generate a threefold increase in lymph node cell proliferation) in the LLNA. These data provide additional evidence that the LLNA is able to discriminate skin sensitizers from chemicals that do not possess a significant skin sensitization potential and is thus a useful method for hazard identification. In addition, this method also offers important animal welfare benefits and may also be useful for risk assessment purposes.