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Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests
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Toxicology in Vitro 21 (2007) 1298–1303 www.elsevier.com/locate/toxinvit
Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests K. Kejlova´ a
a,*
, D. Jı´rova´ a, H. Bendova´ a, H. Kanda´rova´ b, Z. Weidenhoffer a, H. Kola´rˇova´ c, M. Liebsch b
National Reference Center for Cosmetics, National Institute of Public Health, Sˇroba´rova 48, 100 42 Prague 10, Czech Republic b Federal Institute for Risk Assessment (BfR), ZEBET at the BfR, Berlin, Germany c Medical Faculty of Palacky´ University, Olomouc, Czech Republic Received 29 September 2006; accepted 29 May 2007 Available online 13 June 2007
Abstract The aim of this study was to clarify the differences in the phototoxicity of bergamot oil obtained from four different suppliers. Spectral and chemical analyses were performed to identify presence of photoactive compounds in the test samples. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D PT). Confirmatory photopatch tests in a group of volunteers were performed using the first non-phototoxic concentration determined in the H3D PT. The spectral and chemical analyses revealed, that two samples of bergamot oil exhibited a potential for photoactivation. These oils were subsequently classified as phototoxic in the 3T3 NRU PT, however, only on the basis of borderline results and depending on the solvent used. H3D PT revealed clear classifications, correlating well with the findings of spectral and chemical analysis. The test was, however, not yet capable of precise prediction of safe, non-phototoxic concentrations. Additional endpoints, e.g. interleukin determination might be employed to increase the sensitivity of the test. Although the study showed the usefulness of the tiered testing strategy, currently, the extrapolation of in vitro results to human situation may be performed only to a limited extent. 2007 Elsevier Ltd. All rights reserved. Keywords: Phototoxicity; Bergamot oils; 3T3 NRU phototoxicity test; 3D human skin model; Photopatch test
1. Introduction Bergamot oil (BO) is a widely used aromatic (fragrance) ingredient in cosmetics that may be applied on sun-exposed skin areas, although some components of bergamot oil (bergapten, citropten, bergamoten and other furocoumarins) may cause phototoxic effects (Tisserand and Balacs, 1995). The International Fragrance Association (IFRA, 1992) recommends a maximum of 0.4% of bergamot oil Abbreviations: BO, bergamot oil; 3T3 NRU PT, 3T3 neutral red uptake phototoxicity test; PIF, photoirritation factor; MPE, mean photoeffect; H3D PT, reconstructed human 3D skin model phototoxicity test. * Corresponding author. Tel.: +420 267082327; fax: +420 267082386. E-mail address: [email protected] (K. Kejlova´). 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.05.016
in the final leave-on products for application to areas of skin exposed to sunshine to avoid phototoxic and photocarcinogenic hazard. In order to guarantee safety, it is suggested to remove bergapten and other phototoxic components by distillation, the resulting oil being known as bergamot FCF (furocoumarin-free). The aim of this study was not only to identify possible differences in the phototoxicity of bergamot oils obtained from various suppliers with different content of photoactive components, but also to assess the potency of phototoxicity depending on different solvents used according to the current methodology of phototoxicity tests (Spielmann et al., 2000). The test substances were subject to spectral and chemical analyses in order to identify photoactive components present in the individual bergamot oils.
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
The phototoxicity of the samples was evaluated in vitro using the validated 3T3 NRU phototoxicity test (PT) (Commission Directive 2000/33/EC, 2000) and the phototoxicity test on reconstructed human skin model EpiDermTM (Liebsch et al., 1999). On the basis of the EpiDerm phototoxicity assay results, the photopatch testing in a limited group of human volunteers was performed in vivo. In the frame of the Phototoxicity Feasibility Study sponsored by ECVAM, the advantages of the tiered testing strategy using in vitro and in vivo assays were assessed. 2. Materials and methods The tiered testing strategy proceeded as follows: spectral analysis, chemical analysis, in vitro phototoxicity test (PT) in cell culture (3T3 NRU PT), in vitro PT in human skin tissue culture (H3D), in vivo confirmation in a group of volunteers (skin photopatch test). Chemicals: Bergamot oil (CAS No. 8007-75-8) samples were supplied by AROMA s.r.o. (Czech Republic), BIOMEDICA s.r.o. (Czech Republic), SIGMA AG (Germany) and SCHUPP GmbH (Germany). Spectral analysis: Absorbance in the UV–Vis range was determined by means of Spectrophotometer Varian Cary 1E according to OECD Test Guideline 101 (OECD, 1981). Chemical analysis: Analyses of diluted oils were performed by capillary gas chromatography (Restek RTX-1 F&F: 30 m · 0.32 mm i.d. · 0.5 lm d.f.) coupled to a mass spectrometer Fisons Trio 1000. Oven temperature programmed from 35 to 300 C, splitless injection, helium as a carrier, EI with 70 eV. Source of irradiation: The UV light source used in all of the in vitro and in vivo experiments was a doped mercurymetal halide lamp (SOL 500, Dr. Ho¨nle, Germany) which simulates the spectral distribution of natural sunlight. A spectrum almost devoid of UVB (<320 nm) was achieved by filtering with 50% transmission at a wavelength of 335 nm (Filter H1, Dr. Ho¨nle, Germany). The emitted energy was measured before each experiment with a calibrated UVA meter (Type No. 37, Dr. Ho¨nle, Germany). 2.1. Methods in vitro The 3T3 Neutral Red Uptake Phototoxicity Test was performed according to INVITTOX Protocol No. 78 (Liebsch and Spielmann, 1998), using 3T3 Balb/c fibroblasts (L1, ECACC No. 86052701), passage 60–85. For concentration–response analysis Phototox Version 2.0 software (obtained from ZEBET, Germany) was employed. A test substance is predicted as having a potential phototoxic hazard if the photoirritation factor (PIF), calculated as the ratio of toxicity for each substance with and without UV light, is higher than 5 (Commission Directive 2000/33/ EC, 2000; Spielmann et al., 1998). Using the Phototox software, a second predictor of phototoxicity, the mean photo effect (MPE) was also calculated. The MPE is a statistical comparison of the dose–response curves obtained with
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and without UV and a test substance is predicted as phototoxic if MPE is higher than 0.1 (Holzhu¨tter, 1997). According to the OECD Test Guideline 432, a test substance with a PIF >2 and <5 or an MPE >0.1 and <0.15 is predicted as ‘‘probably phototoxic’’ (OECD, 2004). The EpiDerm Skin Phototoxicity Test was conducted according to Liebsch et al. (1999). 3D skin models, EpiDerm EPI-200 (0.63 cm2), were supplied by MatTek, USA. Before dosing the tissues were preincubated in fresh medium for 1 h (37C, 5% CO2). The test materials diluted in distilled water or sesame oil were applied overnight (16– 20 h) in a volume of 50 ll per tissue in water and 20 ll per tissue in oil. One set of tissues was irradiated with a nontoxic dose of 6 J/cm2 (as measured in the UVA range). One day after the treatment and UVA exposure the cytotoxicity was detected as reduction of mitochondrial conversion of MTT to formazan. The optical density of the formazan extract was determined at 540 nm by means of Spectrophotometer Varian Cary 1E. The results of mean tissue values in the presence and absence of UV light were compared and a test substance was considered to be phototoxic, if one or more test concentrations of the (+UVA) part of the experiment revealed a decrease in viability exceeding 30% when compared with identical concentrations of the ( UVA) part of the experiment (Liebsch et al., 1997). 2.2. Method in vivo The photopatch test in human volunteers was performed according to Neumann et al. (2000) in a limited group of 5 healthy females, aged 27–62. Test concentrations were selected according to the EpiDerm phototoxicity test results, i.e. for each bergamot oil the highest non-phototoxic/non-cytotoxic concentration of the samples diluted in water and sesame oil was employed. The test samples were applied in occlusion (Finn Chamber, USA), using saturated filter paper discs (diameter of 10 mm), on the lower back in two areas on both sides of the spine. The exposure time was 1 h and immediately after removal of the patch test the irradiation of one test area (at a dose of 5 J/cm2, as measured in the UVA range) was performed. The other non-irradiated area served as a control. Test reactions were recorded 24 h, 48 h and 72 h after irradiation. Any reaction (erythema, oedema or pigmentation) was recorded and expressed as ratio of positivity. The selection of volunteers and the test method were carried out in compliance with the ethical principles of the Declaration of Helsinki and subsequent revisions (CIOMS, 2002). The study was approved by the Ethical Review Committee of the National Institute of Public Health, Prague. 3. Results The spectrophotometric analysis in UV–Vis range revealed higher absorbance (in the range of 300–360 nm)
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
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in case of BO SCHUPP and BO SIGMA, compared to BO AROMA and BO BIOMEDICA (Fig. 1). Chemical analysis enabled identification and quantification of photoactive compounds present in the test samples (Table 1). A higher content of photoactive compounds present in BO SIGMA and BO SCHUPP was detected. Besides bergapten, differences in other potentially phototoxic components content, as citropten, bergamoten, geranial and neral, were identified. Amongst the four test samples of bergamot oil, two oils were classified phototoxic and two were classified non-phototoxic in the 3T3 NRU PT, however, only on the basis of borderline phototoxicity results. Moreover, the test results were highly dependent on the solvent used for the test samples dilution (Table 2). The following in vitro Epiderm Skin Phototoxicity Test identified as samples with potential of phototoxicity BO AROMA in oil, BO SIGMA in both water and sesame oil and BO SCHUPP in water (Fig. 2a–h). No phototoxicity was proved for BO AROMA in water and BO BIOMEDICA in both water and sesame oil. For technical reasons the phototoxicity of BO SCHUPP in sesame oil was tested only in a limited range of dilutions; up to the concentration of 0.1% no phototoxicity was observed. Subsequent human in vivo photopatch tests employed the highest non-phototoxic/non-cytotoxic concentrations
Table 2 Bergamot oils phototoxicity – 3T3 NRU PT Bergamot oil
Solvent
Run
PIF
AROMA
Ethanol (1% in PBS)
1 2 1 2 1 2
1.218 1.067 1.006 1.185 1.075 0.905
0.027 0.004 0.005 0.038 0.004 0.064
1 2 1 2 1 2
1.377 1.178 1.051 0.904 1.310 1.113
0.004 0.016 0.003 0.017 0.113a 0.031
1 2 1 2 1 2
1.159 1.383 1.266 1.666 1.648 2.380a
0.000 0.147a 0.048 0.144a 0.017 0.232b
1 2 1 2 1 2
1.126 1.358 1.419 1.410 2.581a 3.415a
0.126a 0.156b 0.081 0.088 0.289b 0.348b
DMSO (1% in PBS) PBS BIOMEDICA
DMSO (1% in PBS) PBS SIGMA
PBS SCHUPP
BO AROMA
Absorbance
0.14 0.12
Ethanol (1% in PBS) DMSO (1% in PBS) PBS
a
Probably phototoxic. Phototoxic (according to OECD TG 432).
obtained in the EpiDerm Skin Phototoxicity Test. From all the tested samples, the potential of phototoxicity was identified in vivo only for BO SIGMA and BO SCHUPP diluted in water (Table 3). The early erythema reaction gradually developed into persistent pigmentation during a 7 day period. An example of skin reactions developed in case of one volunteer 48 h after irradiation is shown in Fig. 3.
0.18 0.16
Ethanol (1% in PBS) DMSO (1% in PBS)
b
0.2
Ethanol (1% in PBS)
MPE
BO SCHUPP
0.1 0.08 BO SIGMA
0.06
4. Discussion
0.04 0.02
BO BIOMEDICA
0 280
300
320 340 360 Wavelength [nm]
380
400
Fig. 1. Bergamot oils – spectral analysis. The absorption spectra of BO AROMA (- Æ -), BO BIOMEDICA (– – –), BO SIGMA (Æ Æ Æ) and BO SCHUPP (—), solved in DMSO.
Table 1 Characterisation of bergamot oils used in the study using GC chromatography Content of main photoactive components (%) Bergamot oil
Bergapten
Citropten
Bergamoten
Geranial
Neral
AROMA BIOMEDICA SIGMA SCHUPP
n.d. n.d. 0.13 0.18
n.d. n.d. 0.13 0.19
n.d. n.d. 0.86 0.83
n.d. n.d. 0.04 n.d.
n.d. n.d. 0.48 0.43
n.d. – not detected.
It is known that several substances predicted as phototoxic by the 3T3 NRU PT are not phototoxic, when topically applied on skin in low concentrations. This effect is linked to their limited bio-availability in the skin. It is assumed, that the bio-availability of the substances is the key factor for skin reaction development after irradiation (Liebsch et al., 1997; Jones et al., 2003; Jı´rova´ et al., 2005; Liebsch et al., 2005). Since the reconstructed human skin models closely resemble the native human epidermis (Liebsch et al., 1997, 2005) due to the presence of a barrier function similar to the barrier function of human epidermis, the reconstructed human skin models are proposed as an additional tool for verification of positive results of the 3T3 NRU PT and/or for testing of substances incompatible with the 3T3 NRU PT. In contrast to the cell cultures, such as the mouse fibroblasts used in the 3T3 NRU PT, human skin models permit topical application of various types of chemicals and preparations and have
120
UV UV +
100 80 60 40 20 0 0.032
0.100
0.316
1.000
3.160
1301
Bergamot oil SIGMA (solvent - water)
MTT (% of untreated control)
Bergamot oil AROMA (solvent - water)
120
UV UV +
100 80 60 40 20 0
0.032
0.100
0.316
1.000
3.160
[%]
Bergamot oil AROMA (solvent - sesame oil) 120
UV UV +
100 80 60 40 20 0 0.100
0.316
1.000
3.160
MTT (% of untreated control)
[%] Bergamot oil SIGMA (solvent - sesame oil)
UV UV +
120 100 80 60 40 20 0 0.100
10.000
0.316
1.000
3.160
10.000
[%]
Bergamot oil BIOMEDICA (solvent - water) 120
UV UV +
100 80 60 40 20
MTT (% of untreated control)
[%] Bergamot oil SCHUPP (solvent - water) 120
0.032
0.100
0.316
1.000
Bergamot oil BIOMEDICA (solvent - sesame oil) 120
80 60 40 20 0 0.032
UV UV +
60 40 20 0 1.000
3.160
0.316
1.000
3.160
[%]
[%]
80
0.316
0.100
3.160
100
0.100
UV UV +
100
0
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
Bergamot oil SCHUPP (solvent - sesame oil) 120
UV UV +
100 80 60 40 20 0 0.032
0.100
[%]
10.000
[%] Fig. 2. Bergamot oils phototoxicity – H3D PT. Each column represents the mean viability of EpiDerm tissues (n = 2) in the presence (h) and absence (j) of UV light.
less limitations concerning solubility problems. The test materials can be applied to the reconstructed human skin models undiluted, at extreme pH values or even as insoluble materials (Liebsch et al., 2005).
The known prerequisite to a substance phototoxicity is its ability to absorb light energy within the sunlight region (Commission Directive 2000/33/EC, 2000; OECD, 2004). This study proved, that the phototoxic effects of the four
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
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Table 3 Bergamot oils phototoxicity – human photopatch test
1 2 3 4 5 6 7 8
Bergamot oil
Number of positive responses at different time intervals after irradiation 24 h
48 h
72 h
AROMA (1% in water) BIOMEDICA (1% in water) SIGMA (0.1% in water) SCHUPP (0.0316% in water) AROMA (1% in oil) BIOMEDICA (10% in oil) SIGMA (0.316% in oil) SCHUPP (0.1% in oil)
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
1
2
3
4
control water
5
6
7
8
control oil
Fig. 3. Bergamot oils phototoxicity – example of skin reactions 48 h after irradiation. (1) BO AROMA (1% in water); (2) BO BIOMEDICA (1% in water); (3) BO SIGMA (0.1% in water); (4) BO SCHUPP (0.0316% in water); (5) BO AROMA (1% in oil); (6) BO BIOMEDICA (10% in oil); (7) BO SIGMA (0.316% in oil); (8) BO SCHUPP (0.1% in oil).
tested samples of bergamot oil detected in vitro and in vivo in human volunteers were related to their UVA photoabsorption and content of phototoxic components, namely furocoumarins, providing an appropriate solvent was used. BO BIOMEDICA exhibited negligible absorption in the wavelength of interest (300–400 nm), i.e. the UVA and longer UVB light. For this substance, no phototoxicity was detected up to the highest concentration tested using either in vitro or in vivo tests. Similar results were obtained for BO AROMA. Only in the highest concentration tested in the H3D PT assay (3.16% in sesame oil) was revealed phototoxic response. This result may be related either to the differences in experimental tissue batches, or impurities present in the BO AROMA, indicated by the increase in the absorption spectrum around 280–290 nm (see Fig. 1). BO SIGMA and BO SCHUPP exhibited an absorption peak at 300–340 nm, which indicates a potential for photoactivation. Indeed, the phototoxicity of these substances was detected by in vitro methods, however, significantly depending on the solvent used in the assay. In general,
3T3 NRU PT experiments performed using solvents (PBS with addition of 1% ethanol or 1% DMSO) recommended in the official methodologies (Commission Directive 2000/ 33/EC, 2000; OECD, 2004) revealed only ambiguous borderline phototoxicity values for the two samples with potential for photoactivation. Using aqueous dilutions (PBS in case of 3T3 NRU PT and water in case of H3D PT), a clear phototoxic classification was obtained in both in vitro systems. In addition, DMSO when used as a solvent may even attenuate the phototoxic effect of distinct substances as it is a typical hydroxyl radical scavenger, reported to prevent cell damage induced by UVB irradiation (Masaki et al., 1995). Hydroxyl radicals are created after irradiation of photoactive compounds of bergamot oil (e.g. furocoumarins) and are responsible for membrane damage and oxidative chain reactions. Therefore, it may be suggested, that even substances as essential oils with limited water solubility should be tested in aqueous dilutions without the presence of solubilizers as DMSO, to avoid underpredictive classifications due to the radical scavenger effect. During the dermatological test performed in a limited group of human volunteers, the non-phototoxic/non-cytotoxic concentrations of the bergamot oils determined by the H3D PT were employed. The selection of concentrations for human photopatch testing (the highest non-phototoxic in the H3D PT) was based on previous knowledge on the higher permeability of the skin tissues compared to human skin in vivo (Perkins et al., 1996; Ponec, 1992; Ponec et al., 2001, 2002), expecting a lower bio-availability due to more powerful human skin barrier. While the highest non-phototoxic concentrations of all bergamot oils diluted in sesame oil and determined by the H3D PT correlated well with the results of human photopatch tests, aqueous solutions revealed higher phototoxicity than initially expected. Twenty-four hours after irradiation, the skin areas exposed to BO AROMA and SCHUPP revealed erythema, which turned out after 72 h into well developed (tanned-like) pigmentation. The higher human skin reactivity in vivo may be related to additional low doses of UVB emitted by the solar simulator, that is during the experiments in vitro avoided. The irradiation of cells or tissues is performed through the polystyrene lid of the culture plates. This may cause an additional filter effect and thus decrease the final UVB dose (Liebsch and Spielmann, 1998). The tiered testing strategy for the assessment of phototoxicity, comprising physical, chemical, in vitro and in vivo tests, showed its efficacy in the above presented study. Two phototoxic and two non-phototoxic bergamot oils were identified using available methods. Significant influence of the solvent on the final phototoxic effects of the tested substance has been recognised in the 3T3 NRU PT and H3D PT. Although the H3D PT represents a useful tool for confirmation of non-phototoxic and phototoxic samples identified by the 3T3 NRU PT, the extrapolation of in vitro results to the human situation may be performed
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
only to a limited extent. Additional endpoints, e.g. analyses of cytokines release into the culture media, may help to increase the sensitivity of the assay and thus avoid underpredictions. Acknowledgements The study was supported by ECVAM (Contract No. 1986-2002-09 F1 EDISP DE) and by a grant project of the Ministry of Education of the Czech Republic (MSM No. 6198959216). References Commission Directive 2000/33/EC of 25 April 2000. Adapting to technical progress for the 27th time Council Directive 67/548 E on the classification, packaging and labelling of dangerous substances. Annex V method B41 Phototoxicity – In vitro 3T3 NRU Phototoxicity Test. Official Journal of the European Communities L136, pp. 98–107. Council for International Organizations of Medical Sciences, 2002. International Ethical Guidelines for Biomedical Research Involving Human Subjects. Prepared by the Council for International Organizations of Medical Sciences (CIOMS) in collaboration with the World Health Organization (WHO), Geneva. Holzhu¨tter, H.G., 1997. A general measure of in vitro phototoxicity derived from pairs of dose–response curves and its use for predicting the in vivo phototoxicity of chemicals. ATLA 25, 445–462. International Fragrance Association, 1992. Code of Practice. Latest amendments, June 1992. International Fragrance Association, Geneva. Jı´rova´, D., Kejlova´, K., Bendova´, H., Ditrichova´, D., Mezula´nı´kova´, M., 2005. Phototoxicity of bituminous tars—correspondence between results of 3T3 NRU PT, 3D skin model and experimental human data. Toxicology In Vitro 19, 931–934. Jones, P.A., King, A.V., Earl, L.K., Lawrence, R.S., 2003. An assessment of the phototoxic hazard of a personal product ingredient using in vitro assays. Toxicology In Vitro 17, 471–480. Liebsch, M., Barrabas, C., Traue, T., Spielmann, H., 1997. Entwicklung eines in vitro Tests auf dermale Phototoxizita¨t in einem Modell menschlicher Epidermis (EpiDermTM). ALTEX 14, 165–174. Liebsch, M., Spielmann, H., 1998. INVITTOX Protocol No. 78: 3T3 NRU Phototoxicity Assay. European Commission DG-JRC, ECVAM, SIS Database, Last update October 2002.. Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Gerberick, G.F., Cruse, L., Diembeck, W., Pfannenbecker, U., Spieker, J., Holzhu¨tter, H.-G., Brantom, P., Aspin, P., Southee, J., 1999. Prevalidation of the
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epidermTM phototoxicity test. In: Clark, D.G., Lisansky, S.G., Macmillan, R. (Eds.), Alternatives to Animal Testing II. COLIPA, Brussels, pp. 160–167. Liebsch, M., Spielmann, H., Pape, W., Krul, C., Deguercy, A., Eskes, Ch., 2005. UV-Induced Effects. ATLA 33 (1), 131–146. Masaki, H., Atsumi, T., Sakurai, H., 1995. Detection of hydrogen peroxide and hydroxyl radicals in murine skin fibroblasts under UVB irradiation. Biochemical and Biophysical Research Communications 206 (2), 474–479. Neumann, N.J., Ho¨lzle, E., Plewig, G., Schwarz, T., Panizzon, R.G., Breit, R., Ruzicka, T., 2000. Photopatch testing: the 12-year experience of the German, Austrian, and Swiss photopatch test group. Journal of American Academy of Dermatology (February), 183–192. OECD, 1981.OECD Guidelines for the Testing of Chemicals Test No. 101: UV–Vis Absorption Spectra (Original Guideline, adopted 12th May 1981).
Toxicology in Vitro 21 (2007) 1298–1303 www.elsevier.com/locate/toxinvit
Phototoxicity of bergamot oil assessed by in vitro techniques in combination with human patch tests K. Kejlova´ a
a,*
, D. Jı´rova´ a, H. Bendova´ a, H. Kanda´rova´ b, Z. Weidenhoffer a, H. Kola´rˇova´ c, M. Liebsch b
National Reference Center for Cosmetics, National Institute of Public Health, Sˇroba´rova 48, 100 42 Prague 10, Czech Republic b Federal Institute for Risk Assessment (BfR), ZEBET at the BfR, Berlin, Germany c Medical Faculty of Palacky´ University, Olomouc, Czech Republic Received 29 September 2006; accepted 29 May 2007 Available online 13 June 2007
Abstract The aim of this study was to clarify the differences in the phototoxicity of bergamot oil obtained from four different suppliers. Spectral and chemical analyses were performed to identify presence of photoactive compounds in the test samples. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D PT). Confirmatory photopatch tests in a group of volunteers were performed using the first non-phototoxic concentration determined in the H3D PT. The spectral and chemical analyses revealed, that two samples of bergamot oil exhibited a potential for photoactivation. These oils were subsequently classified as phototoxic in the 3T3 NRU PT, however, only on the basis of borderline results and depending on the solvent used. H3D PT revealed clear classifications, correlating well with the findings of spectral and chemical analysis. The test was, however, not yet capable of precise prediction of safe, non-phototoxic concentrations. Additional endpoints, e.g. interleukin determination might be employed to increase the sensitivity of the test. Although the study showed the usefulness of the tiered testing strategy, currently, the extrapolation of in vitro results to human situation may be performed only to a limited extent. 2007 Elsevier Ltd. All rights reserved. Keywords: Phototoxicity; Bergamot oils; 3T3 NRU phototoxicity test; 3D human skin model; Photopatch test
1. Introduction Bergamot oil (BO) is a widely used aromatic (fragrance) ingredient in cosmetics that may be applied on sun-exposed skin areas, although some components of bergamot oil (bergapten, citropten, bergamoten and other furocoumarins) may cause phototoxic effects (Tisserand and Balacs, 1995). The International Fragrance Association (IFRA, 1992) recommends a maximum of 0.4% of bergamot oil Abbreviations: BO, bergamot oil; 3T3 NRU PT, 3T3 neutral red uptake phototoxicity test; PIF, photoirritation factor; MPE, mean photoeffect; H3D PT, reconstructed human 3D skin model phototoxicity test. * Corresponding author. Tel.: +420 267082327; fax: +420 267082386. E-mail address: [email protected] (K. Kejlova´). 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.05.016
in the final leave-on products for application to areas of skin exposed to sunshine to avoid phototoxic and photocarcinogenic hazard. In order to guarantee safety, it is suggested to remove bergapten and other phototoxic components by distillation, the resulting oil being known as bergamot FCF (furocoumarin-free). The aim of this study was not only to identify possible differences in the phototoxicity of bergamot oils obtained from various suppliers with different content of photoactive components, but also to assess the potency of phototoxicity depending on different solvents used according to the current methodology of phototoxicity tests (Spielmann et al., 2000). The test substances were subject to spectral and chemical analyses in order to identify photoactive components present in the individual bergamot oils.
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
The phototoxicity of the samples was evaluated in vitro using the validated 3T3 NRU phototoxicity test (PT) (Commission Directive 2000/33/EC, 2000) and the phototoxicity test on reconstructed human skin model EpiDermTM (Liebsch et al., 1999). On the basis of the EpiDerm phototoxicity assay results, the photopatch testing in a limited group of human volunteers was performed in vivo. In the frame of the Phototoxicity Feasibility Study sponsored by ECVAM, the advantages of the tiered testing strategy using in vitro and in vivo assays were assessed. 2. Materials and methods The tiered testing strategy proceeded as follows: spectral analysis, chemical analysis, in vitro phototoxicity test (PT) in cell culture (3T3 NRU PT), in vitro PT in human skin tissue culture (H3D), in vivo confirmation in a group of volunteers (skin photopatch test). Chemicals: Bergamot oil (CAS No. 8007-75-8) samples were supplied by AROMA s.r.o. (Czech Republic), BIOMEDICA s.r.o. (Czech Republic), SIGMA AG (Germany) and SCHUPP GmbH (Germany). Spectral analysis: Absorbance in the UV–Vis range was determined by means of Spectrophotometer Varian Cary 1E according to OECD Test Guideline 101 (OECD, 1981). Chemical analysis: Analyses of diluted oils were performed by capillary gas chromatography (Restek RTX-1 F&F: 30 m · 0.32 mm i.d. · 0.5 lm d.f.) coupled to a mass spectrometer Fisons Trio 1000. Oven temperature programmed from 35 to 300 C, splitless injection, helium as a carrier, EI with 70 eV. Source of irradiation: The UV light source used in all of the in vitro and in vivo experiments was a doped mercurymetal halide lamp (SOL 500, Dr. Ho¨nle, Germany) which simulates the spectral distribution of natural sunlight. A spectrum almost devoid of UVB (<320 nm) was achieved by filtering with 50% transmission at a wavelength of 335 nm (Filter H1, Dr. Ho¨nle, Germany). The emitted energy was measured before each experiment with a calibrated UVA meter (Type No. 37, Dr. Ho¨nle, Germany). 2.1. Methods in vitro The 3T3 Neutral Red Uptake Phototoxicity Test was performed according to INVITTOX Protocol No. 78 (Liebsch and Spielmann, 1998), using 3T3 Balb/c fibroblasts (L1, ECACC No. 86052701), passage 60–85. For concentration–response analysis Phototox Version 2.0 software (obtained from ZEBET, Germany) was employed. A test substance is predicted as having a potential phototoxic hazard if the photoirritation factor (PIF), calculated as the ratio of toxicity for each substance with and without UV light, is higher than 5 (Commission Directive 2000/33/ EC, 2000; Spielmann et al., 1998). Using the Phototox software, a second predictor of phototoxicity, the mean photo effect (MPE) was also calculated. The MPE is a statistical comparison of the dose–response curves obtained with
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and without UV and a test substance is predicted as phototoxic if MPE is higher than 0.1 (Holzhu¨tter, 1997). According to the OECD Test Guideline 432, a test substance with a PIF >2 and <5 or an MPE >0.1 and <0.15 is predicted as ‘‘probably phototoxic’’ (OECD, 2004). The EpiDerm Skin Phototoxicity Test was conducted according to Liebsch et al. (1999). 3D skin models, EpiDerm EPI-200 (0.63 cm2), were supplied by MatTek, USA. Before dosing the tissues were preincubated in fresh medium for 1 h (37C, 5% CO2). The test materials diluted in distilled water or sesame oil were applied overnight (16– 20 h) in a volume of 50 ll per tissue in water and 20 ll per tissue in oil. One set of tissues was irradiated with a nontoxic dose of 6 J/cm2 (as measured in the UVA range). One day after the treatment and UVA exposure the cytotoxicity was detected as reduction of mitochondrial conversion of MTT to formazan. The optical density of the formazan extract was determined at 540 nm by means of Spectrophotometer Varian Cary 1E. The results of mean tissue values in the presence and absence of UV light were compared and a test substance was considered to be phototoxic, if one or more test concentrations of the (+UVA) part of the experiment revealed a decrease in viability exceeding 30% when compared with identical concentrations of the ( UVA) part of the experiment (Liebsch et al., 1997). 2.2. Method in vivo The photopatch test in human volunteers was performed according to Neumann et al. (2000) in a limited group of 5 healthy females, aged 27–62. Test concentrations were selected according to the EpiDerm phototoxicity test results, i.e. for each bergamot oil the highest non-phototoxic/non-cytotoxic concentration of the samples diluted in water and sesame oil was employed. The test samples were applied in occlusion (Finn Chamber, USA), using saturated filter paper discs (diameter of 10 mm), on the lower back in two areas on both sides of the spine. The exposure time was 1 h and immediately after removal of the patch test the irradiation of one test area (at a dose of 5 J/cm2, as measured in the UVA range) was performed. The other non-irradiated area served as a control. Test reactions were recorded 24 h, 48 h and 72 h after irradiation. Any reaction (erythema, oedema or pigmentation) was recorded and expressed as ratio of positivity. The selection of volunteers and the test method were carried out in compliance with the ethical principles of the Declaration of Helsinki and subsequent revisions (CIOMS, 2002). The study was approved by the Ethical Review Committee of the National Institute of Public Health, Prague. 3. Results The spectrophotometric analysis in UV–Vis range revealed higher absorbance (in the range of 300–360 nm)
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
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in case of BO SCHUPP and BO SIGMA, compared to BO AROMA and BO BIOMEDICA (Fig. 1). Chemical analysis enabled identification and quantification of photoactive compounds present in the test samples (Table 1). A higher content of photoactive compounds present in BO SIGMA and BO SCHUPP was detected. Besides bergapten, differences in other potentially phototoxic components content, as citropten, bergamoten, geranial and neral, were identified. Amongst the four test samples of bergamot oil, two oils were classified phototoxic and two were classified non-phototoxic in the 3T3 NRU PT, however, only on the basis of borderline phototoxicity results. Moreover, the test results were highly dependent on the solvent used for the test samples dilution (Table 2). The following in vitro Epiderm Skin Phototoxicity Test identified as samples with potential of phototoxicity BO AROMA in oil, BO SIGMA in both water and sesame oil and BO SCHUPP in water (Fig. 2a–h). No phototoxicity was proved for BO AROMA in water and BO BIOMEDICA in both water and sesame oil. For technical reasons the phototoxicity of BO SCHUPP in sesame oil was tested only in a limited range of dilutions; up to the concentration of 0.1% no phototoxicity was observed. Subsequent human in vivo photopatch tests employed the highest non-phototoxic/non-cytotoxic concentrations
Table 2 Bergamot oils phototoxicity – 3T3 NRU PT Bergamot oil
Solvent
Run
PIF
AROMA
Ethanol (1% in PBS)
1 2 1 2 1 2
1.218 1.067 1.006 1.185 1.075 0.905
0.027 0.004 0.005 0.038 0.004 0.064
1 2 1 2 1 2
1.377 1.178 1.051 0.904 1.310 1.113
0.004 0.016 0.003 0.017 0.113a 0.031
1 2 1 2 1 2
1.159 1.383 1.266 1.666 1.648 2.380a
0.000 0.147a 0.048 0.144a 0.017 0.232b
1 2 1 2 1 2
1.126 1.358 1.419 1.410 2.581a 3.415a
0.126a 0.156b 0.081 0.088 0.289b 0.348b
DMSO (1% in PBS) PBS BIOMEDICA
DMSO (1% in PBS) PBS SIGMA
PBS SCHUPP
BO AROMA
Absorbance
0.14 0.12
Ethanol (1% in PBS) DMSO (1% in PBS) PBS
a
Probably phototoxic. Phototoxic (according to OECD TG 432).
obtained in the EpiDerm Skin Phototoxicity Test. From all the tested samples, the potential of phototoxicity was identified in vivo only for BO SIGMA and BO SCHUPP diluted in water (Table 3). The early erythema reaction gradually developed into persistent pigmentation during a 7 day period. An example of skin reactions developed in case of one volunteer 48 h after irradiation is shown in Fig. 3.
0.18 0.16
Ethanol (1% in PBS) DMSO (1% in PBS)
b
0.2
Ethanol (1% in PBS)
MPE
BO SCHUPP
0.1 0.08 BO SIGMA
0.06
4. Discussion
0.04 0.02
BO BIOMEDICA
0 280
300
320 340 360 Wavelength [nm]
380
400
Fig. 1. Bergamot oils – spectral analysis. The absorption spectra of BO AROMA (- Æ -), BO BIOMEDICA (– – –), BO SIGMA (Æ Æ Æ) and BO SCHUPP (—), solved in DMSO.
Table 1 Characterisation of bergamot oils used in the study using GC chromatography Content of main photoactive components (%) Bergamot oil
Bergapten
Citropten
Bergamoten
Geranial
Neral
AROMA BIOMEDICA SIGMA SCHUPP
n.d. n.d. 0.13 0.18
n.d. n.d. 0.13 0.19
n.d. n.d. 0.86 0.83
n.d. n.d. 0.04 n.d.
n.d. n.d. 0.48 0.43
n.d. – not detected.
It is known that several substances predicted as phototoxic by the 3T3 NRU PT are not phototoxic, when topically applied on skin in low concentrations. This effect is linked to their limited bio-availability in the skin. It is assumed, that the bio-availability of the substances is the key factor for skin reaction development after irradiation (Liebsch et al., 1997; Jones et al., 2003; Jı´rova´ et al., 2005; Liebsch et al., 2005). Since the reconstructed human skin models closely resemble the native human epidermis (Liebsch et al., 1997, 2005) due to the presence of a barrier function similar to the barrier function of human epidermis, the reconstructed human skin models are proposed as an additional tool for verification of positive results of the 3T3 NRU PT and/or for testing of substances incompatible with the 3T3 NRU PT. In contrast to the cell cultures, such as the mouse fibroblasts used in the 3T3 NRU PT, human skin models permit topical application of various types of chemicals and preparations and have
120
UV UV +
100 80 60 40 20 0 0.032
0.100
0.316
1.000
3.160
1301
Bergamot oil SIGMA (solvent - water)
MTT (% of untreated control)
Bergamot oil AROMA (solvent - water)
120
UV UV +
100 80 60 40 20 0
0.032
0.100
0.316
1.000
3.160
[%]
Bergamot oil AROMA (solvent - sesame oil) 120
UV UV +
100 80 60 40 20 0 0.100
0.316
1.000
3.160
MTT (% of untreated control)
[%] Bergamot oil SIGMA (solvent - sesame oil)
UV UV +
120 100 80 60 40 20 0 0.100
10.000
0.316
1.000
3.160
10.000
[%]
Bergamot oil BIOMEDICA (solvent - water) 120
UV UV +
100 80 60 40 20
MTT (% of untreated control)
[%] Bergamot oil SCHUPP (solvent - water) 120
0.032
0.100
0.316
1.000
Bergamot oil BIOMEDICA (solvent - sesame oil) 120
80 60 40 20 0 0.032
UV UV +
60 40 20 0 1.000
3.160
0.316
1.000
3.160
[%]
[%]
80
0.316
0.100
3.160
100
0.100
UV UV +
100
0
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
MTT (% of untreated control)
K. Kejlova´ et al. / Toxicology in Vitro 21 (2007) 1298–1303
Bergamot oil SCHUPP (solvent - sesame oil) 120
UV UV +
100 80 60 40 20 0 0.032
0.100
[%]
10.000
[%] Fig. 2. Bergamot oils phototoxicity – H3D PT. Each column represents the mean viability of EpiDerm tissues (n = 2) in the presence (h) and absence (j) of UV light.
less limitations concerning solubility problems. The test materials can be applied to the reconstructed human skin models undiluted, at extreme pH values or even as insoluble materials (Liebsch et al., 2005).
The known prerequisite to a substance phototoxicity is its ability to absorb light energy within the sunlight region (Commission Directive 2000/33/EC, 2000; OECD, 2004). This study proved, that the phototoxic effects of the four
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Table 3 Bergamot oils phototoxicity – human photopatch test
1 2 3 4 5 6 7 8
Bergamot oil
Number of positive responses at different time intervals after irradiation 24 h
48 h
72 h
AROMA (1% in water) BIOMEDICA (1% in water) SIGMA (0.1% in water) SCHUPP (0.0316% in water) AROMA (1% in oil) BIOMEDICA (10% in oil) SIGMA (0.316% in oil) SCHUPP (0.1% in oil)
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5
1
2
3
4
control water
5
6
7
8
control oil
Fig. 3. Bergamot oils phototoxicity – example of skin reactions 48 h after irradiation. (1) BO AROMA (1% in water); (2) BO BIOMEDICA (1% in water); (3) BO SIGMA (0.1% in water); (4) BO SCHUPP (0.0316% in water); (5) BO AROMA (1% in oil); (6) BO BIOMEDICA (10% in oil); (7) BO SIGMA (0.316% in oil); (8) BO SCHUPP (0.1% in oil).
tested samples of bergamot oil detected in vitro and in vivo in human volunteers were related to their UVA photoabsorption and content of phototoxic components, namely furocoumarins, providing an appropriate solvent was used. BO BIOMEDICA exhibited negligible absorption in the wavelength of interest (300–400 nm), i.e. the UVA and longer UVB light. For this substance, no phototoxicity was detected up to the highest concentration tested using either in vitro or in vivo tests. Similar results were obtained for BO AROMA. Only in the highest concentration tested in the H3D PT assay (3.16% in sesame oil) was revealed phototoxic response. This result may be related either to the differences in experimental tissue batches, or impurities present in the BO AROMA, indicated by the increase in the absorption spectrum around 280–290 nm (see Fig. 1). BO SIGMA and BO SCHUPP exhibited an absorption peak at 300–340 nm, which indicates a potential for photoactivation. Indeed, the phototoxicity of these substances was detected by in vitro methods, however, significantly depending on the solvent used in the assay. In general,
3T3 NRU PT experiments performed using solvents (PBS with addition of 1% ethanol or 1% DMSO) recommended in the official methodologies (Commission Directive 2000/ 33/EC, 2000; OECD, 2004) revealed only ambiguous borderline phototoxicity values for the two samples with potential for photoactivation. Using aqueous dilutions (PBS in case of 3T3 NRU PT and water in case of H3D PT), a clear phototoxic classification was obtained in both in vitro systems. In addition, DMSO when used as a solvent may even attenuate the phototoxic effect of distinct substances as it is a typical hydroxyl radical scavenger, reported to prevent cell damage induced by UVB irradiation (Masaki et al., 1995). Hydroxyl radicals are created after irradiation of photoactive compounds of bergamot oil (e.g. furocoumarins) and are responsible for membrane damage and oxidative chain reactions. Therefore, it may be suggested, that even substances as essential oils with limited water solubility should be tested in aqueous dilutions without the presence of solubilizers as DMSO, to avoid underpredictive classifications due to the radical scavenger effect. During the dermatological test performed in a limited group of human volunteers, the non-phototoxic/non-cytotoxic concentrations of the bergamot oils determined by the H3D PT were employed. The selection of concentrations for human photopatch testing (the highest non-phototoxic in the H3D PT) was based on previous knowledge on the higher permeability of the skin tissues compared to human skin in vivo (Perkins et al., 1996; Ponec, 1992; Ponec et al., 2001, 2002), expecting a lower bio-availability due to more powerful human skin barrier. While the highest non-phototoxic concentrations of all bergamot oils diluted in sesame oil and determined by the H3D PT correlated well with the results of human photopatch tests, aqueous solutions revealed higher phototoxicity than initially expected. Twenty-four hours after irradiation, the skin areas exposed to BO AROMA and SCHUPP revealed erythema, which turned out after 72 h into well developed (tanned-like) pigmentation. The higher human skin reactivity in vivo may be related to additional low doses of UVB emitted by the solar simulator, that is during the experiments in vitro avoided. The irradiation of cells or tissues is performed through the polystyrene lid of the culture plates. This may cause an additional filter effect and thus decrease the final UVB dose (Liebsch and Spielmann, 1998). The tiered testing strategy for the assessment of phototoxicity, comprising physical, chemical, in vitro and in vivo tests, showed its efficacy in the above presented study. Two phototoxic and two non-phototoxic bergamot oils were identified using available methods. Significant influence of the solvent on the final phototoxic effects of the tested substance has been recognised in the 3T3 NRU PT and H3D PT. Although the H3D PT represents a useful tool for confirmation of non-phototoxic and phototoxic samples identified by the 3T3 NRU PT, the extrapolation of in vitro results to the human situation may be performed
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only to a limited extent. Additional endpoints, e.g. analyses of cytokines release into the culture media, may help to increase the sensitivity of the assay and thus avoid underpredictions. Acknowledgements The study was supported by ECVAM (Contract No. 1986-2002-09 F1 EDISP DE) and by a grant project of the Ministry of Education of the Czech Republic (MSM No. 6198959216). References Commission Directive 2000/33/EC of 25 April 2000. Adapting to technical progress for the 27th time Council Directive 67/548 E on the classification, packaging and labelling of dangerous substances. Annex V method B41 Phototoxicity – In vitro 3T3 NRU Phototoxicity Test. Official Journal of the European Communities L136, pp. 98–107. Council for International Organizations of Medical Sciences, 2002. International Ethical Guidelines for Biomedical Research Involving Human Subjects. Prepared by the Council for International Organizations of Medical Sciences (CIOMS) in collaboration with the World Health Organization (WHO), Geneva. Holzhu¨tter, H.G., 1997. A general measure of in vitro phototoxicity derived from pairs of dose–response curves and its use for predicting the in vivo phototoxicity of chemicals. ATLA 25, 445–462. International Fragrance Association, 1992. Code of Practice. Latest amendments, June 1992. International Fragrance Association, Geneva. Jı´rova´, D., Kejlova´, K., Bendova´, H., Ditrichova´, D., Mezula´nı´kova´, M., 2005. Phototoxicity of bituminous tars—correspondence between results of 3T3 NRU PT, 3D skin model and experimental human data. Toxicology In Vitro 19, 931–934. Jones, P.A., King, A.V., Earl, L.K., Lawrence, R.S., 2003. An assessment of the phototoxic hazard of a personal product ingredient using in vitro assays. Toxicology In Vitro 17, 471–480. Liebsch, M., Barrabas, C., Traue, T., Spielmann, H., 1997. Entwicklung eines in vitro Tests auf dermale Phototoxizita¨t in einem Modell menschlicher Epidermis (EpiDermTM). ALTEX 14, 165–174. Liebsch, M., Spielmann, H., 1998. INVITTOX Protocol No. 78: 3T3 NRU Phototoxicity Assay. European Commission DG-JRC, ECVAM, SIS Database, Last update October 2002.
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epidermTM phototoxicity test. In: Clark, D.G., Lisansky, S.G., Macmillan, R. (Eds.), Alternatives to Animal Testing II. COLIPA, Brussels, pp. 160–167. Liebsch, M., Spielmann, H., Pape, W., Krul, C., Deguercy, A., Eskes, Ch., 2005. UV-Induced Effects. ATLA 33 (1), 131–146. Masaki, H., Atsumi, T., Sakurai, H., 1995. Detection of hydrogen peroxide and hydroxyl radicals in murine skin fibroblasts under UVB irradiation. Biochemical and Biophysical Research Communications 206 (2), 474–479. Neumann, N.J., Ho¨lzle, E., Plewig, G., Schwarz, T., Panizzon, R.G., Breit, R., Ruzicka, T., 2000. Photopatch testing: the 12-year experience of the German, Austrian, and Swiss photopatch test group. Journal of American Academy of Dermatology (February), 183–192. OECD, 1981.OECD Guidelines for the Testing of Chemicals Test No. 101: UV–Vis Absorption Spectra (Original Guideline, adopted 12th May 1981).