Sensitization Properties of Propolis and Balsam of Peru in Guinea Pig Maximization Test (GPMT)

Introduction: Propolis is used in Poland as an active ingredient of some drugs administered externally, dietary supplements and cosmetics. According to the literature, propolis is a non-toxic and safe substance, although it may cause allergic contact dermatitis. Aim: The aim of this study was to assess the allergenic properties of propolis and Balsam of Peru. Material and methods: The study was conducted according to the OECD Guideline for testing of chemi-cals-Skin sensitization with use of Guinea pig maximization test (GPMT). Guinea pigs have similar sensitivity to allergens as human body. Sensitization properties of propolis were tested in comparison with sensitization properties of Balsam of Peru because of the possibility of cross-reaction between those two substances. Results: The skin of guinea pigs in the propolis group showed no visible change compared to the control group during the first (48 h) and second observation (72 h). The skin of guinea pigs from Balsam of Peru group showed discrete erythema in only one case at the first observation (8.33% of the animals). During the second observation, no visible changes were observed compared to the control group. Conclusions: Guinea pig maximization test showed no sensitizing properties of propolis and weak sensitizing properties of Balsam of Peru in concentration of 5%.


Introduction
Propolis is a substance collected by worker bees from the resin of trees, mainly How to cite this paper: Basista-Sołtys, K., from poplar buds and conifer. It is used by them as a multifunctional material in constructing and maintaining hives and also as a protection from microbiological contamination. Propolis is widely used in Poland as an active substance in some drugs administered externally for healing wounds, burns, chilblains, and also as dietary supplements and an ingredient of some cosmetics. The most important properties of propolis are an antibacterial, antivirus and antifungal activity, but also anti-inflammatory and regenerative action [1] [2] [3] [4].
Propolis is known to cause contact allergy, but according to data found in the literature, the hypersensitivity to propolis more often appears in dermatological patients, treated earlier for allergic dermatoses 1.2% -6.7% (average 3.82%) than in healthy population. In healthy population, allergy to propolis seems to be very rare phenomenon (0.64% -1.3%) [5] [6] [7] [8] [9]. Taking into account the data from human population, the data from animal population from the literature is puzzling. The study conducted on guinea pigs by Petersen in 1977 indicated that propolis is a strong allergen because it sensitized 19 out of 25 animals (76% of propolis group) [10]. Today, it occurs that the method use by Petersen-GPMT of Magnusson and Kligman [11] may cause false positive results. This method was criticized by Kligman-one of the authors of this method [12]. Nowadays, for testing skin sensitization, it is recommended to use updated maximization test according to the OECD Guideline for the Testing of Chemicals-Skin sensitization [13]. Moreover, the allergic potential of propolis is very often compared with the allergic potential of Balsam of Peru, because those two substances are cross-reactivity allergens. Data indicate that Balsam of Peru can sensitize two to three times more frequently than propolis [13].

Aim
The aim of the paper is to assess the sensitizing potential of propolis and Balsam of Peru and to compare those two substances.

Ethical Approval
The study was submitted to The First Local Ethical Committee on Animal Testing at the Jagiellonian University in Krakow, Poland. The agreement was approved on the 17 th October, 2012 (number of agreement 142/2012).

Animals
Guinea pigs, all males, weight: 260 -530 g, were procured from Animal Breeding Laboratory number 0055 according to the List of units eligible for breeding experimental animals approved by the Polish Ministry of Science and Higher Education. Animals were acclimatized to the laboratory conditions for 7 days prior to the test. They were kept at constant temperature of 22˚C and 50% of the humidity, under artificial illumination (12 h of light, 12 h of dark) and were nourished and watered ad libitum. They received 4 drops of 100 mg/ml ascorbic acid solution.
Before the study started, guinea pigs were grouped randomly and were weighed. In the propolis group (P) there were 11 animals, in the Balsam of Peru group (B) there were 12 animals and in the control group (C) there were 6 animals. At the beginning the propolis group consisted of 12 animals, but before the study starts one animal was excluded from the study because of its health condition.

The Guinea Pig Maximization test (GPMT)
The test was carried out according to the OECD Guideline for the Testing of Chemicals-Skin sensitization. After the test, the animals were weighed again. Their weights ranged from 362 to 594 g.

Histopathology
Animals were humanely euthanized at the end of the study and the skin samples from the challenge site were collected. Tissue samples were fixed in AFA (alcohol, formalin, and acetic acid) fixative, dehydrated and then routinely embedded in paraffin and sliced in 6 μm thick sections. For histopathological evaluation following stains were performed: hematoxylin and eosin, trichromatic Masson Goldner (TGM), May-Grünwald Giemsa. Samples were examined by light microscopy at magnifications of 20×, 40× and 100× to evaluate the various targets.

Preparation of Blood Smears
Peripheral smears were prepared from a freshly drawn blood samples into S-

Determination of IFN-γ Level
Plasma samples were prepared from the blood collected before animals were euthanized. Blood samples were centrifuged for 10 minutes at 1000× g. The supernatant was collected in a tube and stored at −20˚C. IFN gamma concentration was measured by IFN gamma PIG ELISA kit (ABCAM).

Results
The skin of guinea pigs in propolis group shows no visible changes in compari-  Table 1.
Histopathologically, macroscopic evaluation of the collected parts of the skin showed no changes. The skin was clear, no swelling, blisters or erosions. In the studied parts of the skin there were no skin lesions typical for inflammation.
Neutrophil mobilization was observed mainly in the dermis. In three cases (1 in the propolis group, 2 in the Balsam of Peru group) numerous mast cells in the field of view in the dermis were observed. Most of them were situated below the epidermis and around the capillary (Figure 1(a)) and around the blood vessels in the deeper layers of the dermis (Figure 1(b)).
In other cases, the granulocytes were few or a single in the field of view. Only in one case of the propolis group, numerous neutrophils and monocytes infiltrated epidermis (Figure 2).   Table 2.
The percentage of animals in which mobilization of granulocytes occurred during the GPMT, which may suggest the early stage of an allergic reaction, is shown in Table 3. were below the level of quantification and did not differ between all groups.

Discussion
Although propolis is considered to be a safe and nontoxic substance, there are some reports about sensitizing properties of propolis. Allergic reactions to propolis usually occur as contact dermatitis after topical administration. For assessing the sensitizing potential of a substance it is recommended to apply the Gui-  Additionally, guinea pigs were weighed before and after the study. The loss in weight of 10% or more may indicate systemic toxicity of the substance [15].
During this study, all guinea pigs from each group put weight. The increase in weight ranged from 12.1% to 39.2%, which demonstrates the good health of animals and the lack of toxicity of tested substances. Petersen during the induction exposure for intradermal administration used 5% solution of propolis in propylene glycol and for topical application 50% propolis ethanol solution , which itself may cause skin irritation. As it was shown, irritation threshold for propolis occurs at concentrations higher than 20% [17].
Therefore, the 50% propolis ethanol solution causes strong irritation. It has also been shown that the propylene glycol is permeation enhancer for other substances. One proposed mechanism for it is the modification of the skin barrier by changing the structure of keratin and increasing the concentration of the solution in the stratum corneum [18]. Furthermore, one and the same vehicle should be used for this study to eliminate the risk of causing irritation or sensi-  Table 4.
It should also be noted that in predisposed persons almost any substance may cause contact allergy at high concentrations during the long and frequent exposition [12]. Even anti-inflammatory drugs such as corticosteroids may sensitize some people, particularly if used in inflammatory conditions of the skin [19].
Negative result in the GPMT allows predicting the safety of the substance. Generally, no more testing is needed to determine the possibility of the substance to cause sensitization of the skin. However, there are some exceptions. Nickel is one of the most well-known contact allergens, but in the GPMT it is very hard to induce allergy to nickel [20]. False-negative results, as the one described above, are very rare, but do happen. Just as the reverse case, the substance, which is a potent allergen in the GPMT should not be rejected from the use, especially if it has the advantageous properties [12]. It is always possible to determine the threshold concentration that rarely would sensitize people. 5% propolis ethanol solution does not show sensitization with respect to a healthy adult population.
The problem is, that propolis formulations with much more higher concentration (up to 50%) are present on the market.

Conflict of Interest
Authors report no conflict of interest.