- Email: [email protected]
Evaluation of hypersensitivity to anthraquinone-related substances
Toxicology 193 (2003) 261–267
Evaluation of hypersensitivity to anthraquinone-related substances Seishiro Fujii∗ Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, East Bldg. 149, 13th Street, Charlestown, MA 02129, USA Received 11 April 2003; accepted 23 June 2003
Abstract Evaluation of hypersensitivity to anthraquinone dyes, which are widely used as color additives, was performed in guinea pigs. In animals sensitized with 1% commercial-grade D&C Green No. 6 (Quinizarin Green SS, CI61565), subsequent challenge with the same dye showed a dose-dependent hypersensitivity reaction, whereas challenge with D&C Green No. 6 purified by recrystallization did not. Guinea pigs sensitized with commercial-grade D&C Green No. 6 showed a positive reaction to challenge with quinizarin, an intermediate in dye synthesis, but not to p-toluidine, another intermediate. It was confirmed that quinizarin itself induced hypersensitivity. The cross-reactivity of four kinds of purified dyes, in which the quinizarin contents were 1.2 ppm or less, was evaluated. Purified D&C Green No. 5 (Alizarin Cyanin Green F, CI61570), D&C Green No. 6 and Ext. D&C Violet No. 2 (Alizurol Purple, CI60730) did not produce a hypersensitivity response, while D&C Violet No. 2 (Alizurin Purple SS, CI60725) did. We found that D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 do not intrinsically induce hypersensitivity, and we conclude that the reaction to challenge with the commercial-grade dyes is due to quinizarin contaminating them. In contrast, D&C Violet No. 2 per se induced hypersensitivity. A structure–activity study indicated that 1-hydroxyl and 4-hydroxyl groups in the anthraquinone structure are key factors in hypersensitivity induction by anthraquinone-related compounds. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthraquinone dyes; Quinizarin; Contact hypersensitivity; Cross-sensitivity
1. Introduction There have been many reports on the analysis of tar dyes (Andrisano et al., 1995; Kuo et al., 1998; Tsuji et al., 2000), and on the induction of delayed-type hypersensitivity in guinea pigs and humans by these ∗
Tel.: +1-617-726-7532; fax: +1-617-726-4441. E-mail address: [email protected] (S. Fujii).
dyes (Sugai et al., 1997; Salstad et al., 1994; Seidenari et al., 1997; Ulter et al., 2001, 2002; Seidenari et al., 2002). Among the tar dyes, anthraquinones were reported to induce hypersensitivity in guinea pigs (Hausen and Sawall, 1989) and allergic contact dermatitis in humans exposed to the black ink of a felt-tip marker pen (Miller et al., 1978), or to spectacle frames (Dooms-Goossen et al., 1981), or at a chemical plant (Wilkinson et al., 1994). Photodermatitis due to anthraquinones in humans was also reported (Brandao
0300-483X/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00270-1
262
S. Fujii / Toxicology 193 (2003) 261–267
and Valente, 1988). Aloe has been used since ancient times for medicinal purposes, and is known to contain several naturally existing anthraquinone-related substances, aloe-emodin, chrysophanol, etc. Hypersensitivity to aloe was also reported (Morrow et al., 1980). Four kinds of anthraquinone dyes can be used for drug and cosmetic formulations in the USA as certified colors. One of these colors, D&C Violet No. 2, was reported not to induce hypersensitivity in guinea pigs; however, the methanol extract from a new resorbable monofilament suture containing the dye caused skin sensitization reactions because of quinizarin which was produced by the decomposition of the dye during the melt-spinning process for the production of the suture (Tomihata et al., 1999, 2001). The potential for hypersensitivity to these four dyes and other anthraquinone-related substances has not yet been evaluated systematically. The aim of this study was to investigate the induction of hypersensitivity to anthraquinone dyes and to relate the pattern of cross-sensitization to the chemical structure of anthraquinone-related substances. The results of this study may be relevant to the avoidance of hypersensitivity reactions in humans. Guinea pig assays have been used extensively to detect contact sensitizers, because of their proven value for predicting hypersensitivity reactions to medical products (Vial and Descotes, 1994; Maurer, 1996; Choquet-Kastylevsky and Descotes, 1998; Klecak, 1998). Examples include the guinea pig maximization test (Magnusson and Kligman, 1969), the Buehler assay (Buehler, 1965) and the murine local lymph node assay (Kimber, 2001; van Och et al., 2001). Therefore, a guinea pig assay was used for the present work.
2. Materials and methods 2.1. Materials Four kinds of commercial-grade anthraquinone dyes (D&C Green No. 5, D&C Green No. 6, D&C Violet No. 2 and Ext. D&C Violet No. 2) were obtained from Leeben Colors (presently Sensient Technical Colors, Elmwood Park, NJ, USA). D&C Green No. 6 and D&C Violet No. 2 were purified by recrystallization from toluene. D&C Green No. 5 and Ext. D&C violet No. 2 were purified by precipitation from
aqueous solution with ethyl alcohol after pretreatment of the solution with activated carbon. The chemical structures and contents of quinizarin in these dyes are summarized in Table 1. Quinizarin (Wako Pure Chemical Industries, Osaka, Japan) and hydroxyanthraquinones (Tokyo Kasei Kogyo Co., Tokyo, Japan) were purchased and purified by recrystallization from toluene for the experiments (Table 2). All experiments were performed in Hartley strain female albino guinea pigs (370–420 g). The animals were housed singly in stainless steel wire-mesh cages in a room kept at 23±2 ◦ C and a humidity of 55±5%, and artificially illuminated for 12 h daily. They were fed a standard guinea pig diet (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. 2.2. Methods 2.2.1. Measurement of quinizarin content Quinizarin contents of the dyes were measured with a high-performance liquid chromatography system (Waters Corp., Milford, MA, USA, column: ODS, 4.6 mm i.d. × 250 mm, mobile phase: 1% acetic acid, 4% D.W./methanol, flow rate: 1 ml/min, column oven temperature: 35 ◦ C, detection: UV-Vis at 436 nm). 2.2.2. Evaluation of hypersensitivity reaction All test substances were powdery and unsuitable for the standard guinea pig maximization test because of their poor solubility or suspension properties in water or Freund’s complete adjuvant (FCA), with consequent difficulties in the intradermal injection procedure. Therefore, we used a modified guinea pig testing technique, the adjuvant and patch test, which has equivalent ability to detect hypersensitivity to the guinea pig maximization test (Sato et al., 1981), and is one of the recommended test methods in the guidelines for toxicity studies of drugs in Japan. 2.2.3. Adjuvant and patch test The test procedure followed the reported method (Sato et al., 1981). Briefly, aliquots of 0.1 ml each of a water-in-oil type emulsion (distilled water: FCA = 1:1) were injected intradermally into the four corners of a previously shaved shoulder region (2 cm × 4 cm). At the injection sites, scratches in the shape of a
S. Fujii / Toxicology 193 (2003) 261–267
263
Table 1 List of anthraquinone dyes examined Name
Structure
Quinizarin content Commercial grade (ppm)
D&C Green No. 5 (Alizarin Cyanin Green F, CI61570)
29
Purified grade (ppm)
1.2
440
<1
D&C Violet No. 2 (Alizurin Purple SS, CI60725)
4110
<1
Ext. D&C Violet No. 2 (Alizurol Purple, CI60730)
990
<1
D&C Green No. 6 (Quinizarin Green SS, CI61565)
.
grid were made with the needle used for injection. A closed patch with 0.1 ml of test preparation was applied to the sites for 24 h. Abrasions and sample applications were repeated on the following 2 days. One week after the initial sensitization, 10% sodium lauryl sulfate in petrolatum was applied to the intradermal injection sites. On the next day, a closed patch of the test preparation was applied at the same sites
Table 3 Evaluation of skin reaction (scale for scoring)
Table 2 List of hydroxyanthraquinones examined
Skin evaluation
Usual name
Chemical name
Quinizarin Alizarin Anthrarufin Chrysazin Purpurin Anthrapurpurin
1,4-Dihydroxyanthraquinone 1,2-Dihydroxyanthraquinone 1,5-Dihydroxyanthraquinone 1,8-Dihydroxyanthraquinone 1,2,4-Trihydroxyanthraquinone 1,2,7-Trihydroxyanthraquinone
Anthraquinone:
for 48 h. Two weeks later, a test sample was applied onto the shaved skin of the back. The excess substance was removed from the skin by washing with acetone at 24 h after the challenge application procedure. The skin reactions, fractional response (FR) and mean response (MR), were scored on the basis shown in Table 3 at 1, 24 and 48 h after the washing.
.
Scale for scoring
1. Erythema formation No erythema Very slight erythema Well-defined erythema Moderate to severe erythema Severe erythema with slight eschar formation
0 1 2 3 4
2. Edema formation No edema Slight edema Moderate edema Severe edema
0 1 2 3
Overall maximum score Mean response = n1 [(1) + (2)]/total number of animals.
7
264
S. Fujii / Toxicology 193 (2003) 261–267
Table 4 Contact hypersensitivity of D&C Green No. 6 Challenge substance
D&C Green No. 6 (commercial)
D&C Green No. 6 (purified) Quinizarin p-toluidine
Concentration (%)
1 0.1 0.01 0.001 1 0.1 0.01 1 1
Sensitized group
Control group
FR
MR
FR
MR
8/10 7/10 2/10 0/10 0/10 0/10 0/10 8/10 0/10
2.1 1.4 0.3 0 0 0 0 3.0 0
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
0 0 0 0 0 0 0 0 0
Induction: 1% D&C Green No. 6 (commercial grade). Solvent: acetone, FR: fractional response, MR: mean response.
3. Results The results obtained with commercial-grade and purified D&C Green No. 6 are summarized in Table 4. When guinea pigs were sensitized with 1% commercial-grade D&C Green No. 6, a positive dose-dependent response was obtained to challenge with the commercial-grade dye. However, no response was detected to 1% purified D&C Green No. 6. Quinizarin, a synthetic intermediate of the dye, gave a positive reaction at 1%, while p-toluidine, another intermediate, did not. Next, several commercial-grade and purified anthraquinone dyes were examined for contact hypersensitivity in guinea pigs sensitized with 0.2% quinizarin. As shown in Table 5, three commercial-grade dyes, D&C Green No. 6, D&C Violet No. 2 and Ext. Violet No. 2, gave positive reactions, though purified samples of these three dyes did not. In the case of D&C Green No. 5, neither commercial grade nor purified dye induced any reaction. Quinizarin gave a strong positive reaction even at 0.2%. The results of cross-reactivity testing of the four purified dyes in guinea pigs with 1% purified dyes are shown in Table 6. Purified D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 did not show any positive reaction, though purified D&C Violet No. 2 gave a positive reaction. The results of contact hypersensitivity testing of quinizarin are summarized in Fig. 1. Four different concentrations of quinizarin were used for the in-
Table 5 Cross-reactivity of anthraquinone dyes in guinea pigs sensitized with quinizarin Challenge substance
Concentration (%)
Sensitized group
Control group
FR
MR
FR
MR
D&C Green No. 5 Commercial 1 Purified 1
0/14 0/14
0 0
0/10 0/10
0 0
D&C Green No. 6 Commercial 1 Purified 1
5/14 0/14
1.1 0
0/10 0/10
0 0
D&C Violet No. 2 Commercial 1 Purified 1
8/14 0/14
1.1 0
0/10 0/10
0 0
Ext. D&C Violet No. 2 Commercial 1 Purified 1
1/14 0/14
0.1 0
0/10 0/10
0 0
Quinizarin
14/14
4.6
0/10
0
0.2
Induction: 0.2% quinizarin. Solvent: acetone, FR: fractional response, MR: mean response.
duction and challenge procedures in the experiments. Guinea pigs sensitized with 0.2% quinizarin did not give a positive reaction to challenge with 0.0002%, but positive reactions were confirmed at challenge
Fig. 1. Concentration dependence of contact hypersensitivity to quinizarin in guinea pigs. Four groups of guinea pigs (n = 10 for each group) were sensitized with four different concentrations of quinizarin and challenged with four different concentrations of quinizarin. Control group guinea pigs (n = 10) applied with four different concentrations of quinizarin were negative (FR = 0/10, MR = 0). Solvent: acetone.
S. Fujii / Toxicology 193 (2003) 261–267
265
Table 6 Cross-reactivity of anthraquinone dyes Challenge substance and concentration (%)
Induction substance D&C Green No. 5
D&C Green No. 6
D&C Violet No. 2
Ext. D&C Violet No. 2
FR
MR
FR
MR
FR
MR
FR
MR
Commercial 1 0.1 0.01 0.001
0/10 0/10 0/10 n.d.
0 0 0
0/10 0/10 0/10 n.d.
0 0 0
9/10 7/10 5/10 0/10
1.8 1.7 0.6 0
0/10 0/10 0/10 n.d.
0 0 0
Purified 1 0.1 0.01 0.001
0/10 0/10 0/10 n.d.
0 0 0
0/10 0/10 0/10 n.d.
0 0 0
10/10 10/10 8/10 0/10
2.2 2.0 0.9 0
0/10 0/10 0/10 n.d.
0 0 0
Quinizarin 0.2 0.02
0/10 0/10
0 0
0/10 0/10
0 0
3/10 0/10
0.3 0
0/10 0/10
0 0
Induction: 1% of each anthraquinone dye (purified grade). Solvent: acetone, n.d.: not done, FR: fractional response, MR: mean response. Control group: 1% of each dye and 0.2% of quinizarin were negative (FR = 0/10, MR = 0).
concentrations of 0.002, 0.02 and 0.2%. Guinea pigs induced with 0.0002% quinizarin did not show a positive reaction on challenge with 0.2%. Experiments on cross-reactivity among quinizarin and structurally-related substances were also performed. As shown in Table 7, quinizarin per se and purpurin per se showed positive reactions and these dyes showed cross-reactivity. Alizarin, anthrarufin, chrysazin and anthrapurpurin did not show either positive reaction or cross-reactivity among the evaluated substances.
4. Discussion The results of contact hypersensitivity testing of D&C Green No. 6 (Table 4) and cross-reactivity testing of purified anthraquinone dyes (Table 6) show that commercial-grade D&C Green No. 6 exhibited hypersensitivity reactions, whereas purified D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 did not. The hypersensitivity to the commercialgrade dyes was concluded to be due to quinizarin, which is used in the synthesis of anthraquinone dyes
Table 7 Cross-reactivity of hydroxyanthraquinones Challenge substance
Induction substance Quinizarin
Quinizarin Alizarin Anthrarufin Chrysazin Purpurin Anthrapurpurin
Alizarin
Anthrarufin
Chrysazin
Purpurin
Anthrapurpurin
FR
MR
FR
MR
FR
MR
FR
MR
FR
MR
FR
MR
4/5 0/5 0/5 0/5 4/5 0/5
2.8 0 0 0 2.6 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
4/5 0/5 0/5 0/5 3/5 0/5
2.6 0 0 0 2.0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
Induction: 0.5% each hydroxyanthraquinone in N-methylpyrrolidone. Challenge: 0.03% of each hydroxyanthraquinone in acetone, FR: fractional response, MR: mean response. Control group: 0.03% acetone solution of each substance was negative (FR = 0/5, MR = 0).
266
S. Fujii / Toxicology 193 (2003) 261–267
and is present as an impurity in commercial-grade dyes. In contrast, hypersensitivity to commercial-grade D&C Violet No. 2 appeared to be due to both the dye per se and quinizarin present as an impurity. D&C Violet No. 2 was reported not to be allergenic in the guinea pig maximization test with some modifications by Tomihata et al. (1999). They used a concentration of 5% in olive oil for intradermal injection, 10% in petrolatum for 48 h (closed application) at the induction stage and 10% in acetone for 24 h (open application) at challenge. In contrast, we used 1% in acetone for 24 h (closed application) for 3 consecutive days applied to water-in-oil emulsion (with FCA)-injected sites, 1% in acetone for 48 h (closed application) at the induction stage and maximum 1% in acetone for 24 h (closed application) at challenge. The differences between the reported results by Tomihata et al. (1999) and the results in Table 6 are likely to be due to the differences of experimental methods, especially at the challenge stage, and solvents used for the experiments. The adjuvant and patch test that we used was developed for the detection of weak allergens and to evaluate the allergenicity of materials unsuitable for testing by intradermal injection, and involves the use of FCA to stimulate the immune system of the animal and external application instead of intradermal injection of the test compound in the induction stage. It was reported that test results obtained by using the adjuvant and patch test correlated well with those obtained in the guinea pig maximization test (Sato et al., 1981). Interestingly, purified Ext. D&C Violet No. 2, a hydrophilic form of D&C Violet No. 2, did not induce hypersensitivity. A similar phenomenon was reported in relation to D&C Yellow No. 10 and D&C Yellow No. 11 (Sato et al., 1984). Strong and dose-dependent hypersensitivity to quinizarin was confirmed (Fig. 1). Guinea pigs sensitized with 0.002% quinizarin showed no reaction on challenge with 0.002%, though a weak positive reaction (FR = 1/10, MR = 0.1) was seen at a challenge concentration of 0.02%. These results indicate that guinea pigs could be sensitized with 0.002% quinizarin. Guinea pigs treated with 0.0002% quinizarin showed no positive reaction even to a challenge concentration of 0.2%. These results mean that guinea pigs were not sensitized by exposure to 0.0002% quinizarin. Quinizarin was reported to in-
duce allergic reactions when evaluated at 0.01% for intradermal injection, 10% for closed application in the induction stage and 0.01% for open application at the challenge stage. The results in Fig. 1 indicate stronger allergenicity of quinizarin than was previously reported (Tomihata et al., 2001), but the differences of test methods and solvents used in the experiments are likely to account for this. The reported results were obtained when quinizarin was evaluated at four different concentrations (0.0001–0.1%) in olive oil for intradermal injection, 10% in petrolatum for 48 h (closed application) at the induction stage and five different concentrations (0.0005–1.0%) in acetone for 24 h (open application) at challenge. In the present study, we evaluated the hypersensitivity to quinizarin by using the adjuvant and patch test, involving four different concentrations in acetone for 24 h (closed application) for 3 consecutive days on water-in-oil emulsion injected sites, four different concentrations in acetone for 48 h (closed application) at the induction stage and four different concentrations in acetone for 24 h (closed application) at challenge. The differences in test methods, especially at the challenge stage, and solvents used for the experiments might explain the stronger reaction to quinizarin in our study. In the experiment on cross-reactivity of hydroxyanthraquinones (Table 7), the results showed that 1- and 4-hydroxyl groups of anthraquinone-related compounds play a key role in hypersensitivity induction. It is important to note that anthraquinone-related substances, including some of the evaluated hydroxyanthraquinones, exist in plants. Generally, one group of these compounds has two or three hydroxyl groups on the benzene ring on one side of the molecule, while another group has one or two hydroxyl groups on benzene rings on both sides of the molecule. Examples include alizarin, purpurin, chrysophanol, emodin, aloe-emodin, rhein, etc. in madder, rhubarb and aloe (Okada and Mitsuhashi, 2002). It is known that sennoside, which is not an anthraquinone derivative, but an anthrone derivative, also exists in rhubarb, aloe, etc. A study on hypersensitivity induction by sennoside may be warranted. References Andrisano, V., Cavrini, V., Summer, P., Passuti, S., 1995. Determination of impurities in oxidation hair dyes as raw materials
S. Fujii / Toxicology 193 (2003) 261–267 by liquid chromatography (HPLC). Int. J. Cosmet. Sci. 17, 53–60. Brandao, F.M., Valente, A., 1988. Photodermatitis from anthraquinone. Contact Dermat. 18, 171–172. Buehler, E.V., 1965. Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol. 91, 171–177. Choquet-Kastylevsky, G., Descotes, J., 1998. Value of animal models for predicting hypersensitivity reactions to medicinal products. Toxicology 129, 27–35. Dooms-Goossen, A., Bonamte, A., Parys, M., Dooms, M., 1981. Sensitizing anthraquinone dye in spectacle frames. Contact Dermat. 7, 214–215. Hausen, B.M., Sawall, E.M., 1989. Sensitization experiments with textile dyes in guinea pigs. Contact Dermat. 20, 27–31. Kimber, I., 2001. The local lymph node assay and potential application to the identification of dug allergens. Toxicology 158, 59–64. Klecak, G., 1998. Test methods for allergic contact dermatitis in animals. In: Marzulli, F.N., Maibach, H.I. (Eds.), Dermatotoxicology Methods. Taylor & Francis, Bristol, PA, pp. 121–143. Kuo, K.L., Huang, H.Y., Hsieh, Y.Z., 1998. High-performance capillary electrophoretic analysis of synthetic food colorants. Chromatographia 47, 249–256. Magnusson, B., Kligman, A.M., 1969. The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52, 268–276. Maurer, T., 1996. Guinea pig predictive tests. In: Kimber, I., Maurer, T. (Eds.), Toxicology of Contact Hypersensitivity. Taylor & Francis, Bristol, PA, pp. 107–126. Miller, M.M., Goldgerg, H.S., Wilkerson, W.G., 1978. Allergic contact dermatitis to 1,4-bis(isopropylamino)anthraquinone. Arch. Dermatol. 114, 1793–1794. Morrow, D.M., Rapaport, M.J., Strick, R.A., 1980. Hypersensitivity to aloe. Arch. Dermatol. 116, 1064–1065. Okada, M., Mitsuhashi, H., 2002. Newly Revised Illustrated Medicinal Plants of the World. Hokuryukan Co., Tokyo, pp. 45, 499, 618 and 743. Salstad, D.M., Tepper, J.S., Doerfler, D.L., Qasim, M., Selegrade, M.K., 1994. Evaluation of azo and two anthraquinone dyes for allergic potential. Fundam. Appl. Toxicol. 4, 569–577. Sato, Y., Katsumura, Y., Ichikawa, H., Kobayashi, T., Kozuka, T., Morikawa, F., Ohta, S., 1981. A modified technique of
267
guinea pig testing to identify delayed hypersensitivity allergens. Contact Dermat. 7, 225–237. Sato, Y., Kutsuna, H., Kobayashi, T., Mitsui, T., 1984. D&C Yellow Nos. 10 and 11: chemical composition analysis and delayed contact hypersensitivity testing in the guinea pigs. Contact Dermat. 10, 30–38. Seidenari, S., Montovani, L., Manzini, B.M., Pignatti, M., 1997. Cross-sensitization between azo dyes and para-amino compound. Contact Dermat. 36, 91–96. Seidenari, S., Giusti, F., Massone, F., Mantovani, L., 2002. Sensitization to disperse dyes in a patch test population over a five-year period. Am. J. Contact Dermat. 13, 101–107. Sugai, T., Takahashi, Y., Takagi, T., 1997. Pigmented cosmetic dermatitis and coal tar dyes. Contact Dermat. 5, 249–256. Tomihata, K., Suzuki, M., Sato, H., 1999. Skin sensitization study of a new resorbable suture containing D&C Violet No. 2 on guinea pig maximization test. Jpn. J. Dermatoallergology 7, 101–108. Tomihata, K., Suzuki, M., Sato, H., Kitagawa, M., 2001. Sensitizer contained in heat-decomposed dye. J. Biomed. Mater. Res. 15, 531–539. Tsuji, S., Okada, M., Akamatsu, Y., Tonogai, Y., 2000. Studies on rejected Food Blue No. 1 (Brilliant Blue FCF) Aluminium Lake. Bull. Natl. Inst. Health Sci. 118, 131–134. Ulter, W., Geier, J., Lessmann, H., Hausen, B.M., 2001. Contact allergy to Disperse Blue 106 and Disperse Blue 124 in German and Austrian patients, 1995 to 1999. Contact Dermat. 44, 173– 177. Ulter, W., Lessmann, H., Geier, J., Becker, D., Fucks, T., Richter, G., 2002. The spectrum of allergic (cross-)sensitivity in clinical patch testing with ‘para amino’ compounds. Allergy 57, 319– 322. van Och, F.M.M., Vandebriel, R.J., Prinsen, M.K., De Jong, W.H., Slob, W., van Loveren, H., 2001. Comparison of dose–response of contact allergens using the guinea pig maximization test and the local lymph node assay. Toxicology 167, 207– 215. Vial, T., Descotes, J., 1994. Contact sensitization assays in guinea-pigs: are they predictive of the potential for systemic allergic reactions? Toxicology 93, 63–75. Wilkinson, S.M., Brittain, J., Beck, M.H., 1994. Allergic contact dermatitis from an anthraquinone derivatives in a chemical plant. Contact Dermat. 30, 241–242.
Evaluation of hypersensitivity to anthraquinone-related substances Seishiro Fujii∗ Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, East Bldg. 149, 13th Street, Charlestown, MA 02129, USA Received 11 April 2003; accepted 23 June 2003
Abstract Evaluation of hypersensitivity to anthraquinone dyes, which are widely used as color additives, was performed in guinea pigs. In animals sensitized with 1% commercial-grade D&C Green No. 6 (Quinizarin Green SS, CI61565), subsequent challenge with the same dye showed a dose-dependent hypersensitivity reaction, whereas challenge with D&C Green No. 6 purified by recrystallization did not. Guinea pigs sensitized with commercial-grade D&C Green No. 6 showed a positive reaction to challenge with quinizarin, an intermediate in dye synthesis, but not to p-toluidine, another intermediate. It was confirmed that quinizarin itself induced hypersensitivity. The cross-reactivity of four kinds of purified dyes, in which the quinizarin contents were 1.2 ppm or less, was evaluated. Purified D&C Green No. 5 (Alizarin Cyanin Green F, CI61570), D&C Green No. 6 and Ext. D&C Violet No. 2 (Alizurol Purple, CI60730) did not produce a hypersensitivity response, while D&C Violet No. 2 (Alizurin Purple SS, CI60725) did. We found that D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 do not intrinsically induce hypersensitivity, and we conclude that the reaction to challenge with the commercial-grade dyes is due to quinizarin contaminating them. In contrast, D&C Violet No. 2 per se induced hypersensitivity. A structure–activity study indicated that 1-hydroxyl and 4-hydroxyl groups in the anthraquinone structure are key factors in hypersensitivity induction by anthraquinone-related compounds. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthraquinone dyes; Quinizarin; Contact hypersensitivity; Cross-sensitivity
1. Introduction There have been many reports on the analysis of tar dyes (Andrisano et al., 1995; Kuo et al., 1998; Tsuji et al., 2000), and on the induction of delayed-type hypersensitivity in guinea pigs and humans by these ∗
Tel.: +1-617-726-7532; fax: +1-617-726-4441. E-mail address: [email protected] (S. Fujii).
dyes (Sugai et al., 1997; Salstad et al., 1994; Seidenari et al., 1997; Ulter et al., 2001, 2002; Seidenari et al., 2002). Among the tar dyes, anthraquinones were reported to induce hypersensitivity in guinea pigs (Hausen and Sawall, 1989) and allergic contact dermatitis in humans exposed to the black ink of a felt-tip marker pen (Miller et al., 1978), or to spectacle frames (Dooms-Goossen et al., 1981), or at a chemical plant (Wilkinson et al., 1994). Photodermatitis due to anthraquinones in humans was also reported (Brandao
0300-483X/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0300-483X(03)00270-1
262
S. Fujii / Toxicology 193 (2003) 261–267
and Valente, 1988). Aloe has been used since ancient times for medicinal purposes, and is known to contain several naturally existing anthraquinone-related substances, aloe-emodin, chrysophanol, etc. Hypersensitivity to aloe was also reported (Morrow et al., 1980). Four kinds of anthraquinone dyes can be used for drug and cosmetic formulations in the USA as certified colors. One of these colors, D&C Violet No. 2, was reported not to induce hypersensitivity in guinea pigs; however, the methanol extract from a new resorbable monofilament suture containing the dye caused skin sensitization reactions because of quinizarin which was produced by the decomposition of the dye during the melt-spinning process for the production of the suture (Tomihata et al., 1999, 2001). The potential for hypersensitivity to these four dyes and other anthraquinone-related substances has not yet been evaluated systematically. The aim of this study was to investigate the induction of hypersensitivity to anthraquinone dyes and to relate the pattern of cross-sensitization to the chemical structure of anthraquinone-related substances. The results of this study may be relevant to the avoidance of hypersensitivity reactions in humans. Guinea pig assays have been used extensively to detect contact sensitizers, because of their proven value for predicting hypersensitivity reactions to medical products (Vial and Descotes, 1994; Maurer, 1996; Choquet-Kastylevsky and Descotes, 1998; Klecak, 1998). Examples include the guinea pig maximization test (Magnusson and Kligman, 1969), the Buehler assay (Buehler, 1965) and the murine local lymph node assay (Kimber, 2001; van Och et al., 2001). Therefore, a guinea pig assay was used for the present work.
2. Materials and methods 2.1. Materials Four kinds of commercial-grade anthraquinone dyes (D&C Green No. 5, D&C Green No. 6, D&C Violet No. 2 and Ext. D&C Violet No. 2) were obtained from Leeben Colors (presently Sensient Technical Colors, Elmwood Park, NJ, USA). D&C Green No. 6 and D&C Violet No. 2 were purified by recrystallization from toluene. D&C Green No. 5 and Ext. D&C violet No. 2 were purified by precipitation from
aqueous solution with ethyl alcohol after pretreatment of the solution with activated carbon. The chemical structures and contents of quinizarin in these dyes are summarized in Table 1. Quinizarin (Wako Pure Chemical Industries, Osaka, Japan) and hydroxyanthraquinones (Tokyo Kasei Kogyo Co., Tokyo, Japan) were purchased and purified by recrystallization from toluene for the experiments (Table 2). All experiments were performed in Hartley strain female albino guinea pigs (370–420 g). The animals were housed singly in stainless steel wire-mesh cages in a room kept at 23±2 ◦ C and a humidity of 55±5%, and artificially illuminated for 12 h daily. They were fed a standard guinea pig diet (Oriental Yeast Co., Tokyo, Japan) and water ad libitum. 2.2. Methods 2.2.1. Measurement of quinizarin content Quinizarin contents of the dyes were measured with a high-performance liquid chromatography system (Waters Corp., Milford, MA, USA, column: ODS, 4.6 mm i.d. × 250 mm, mobile phase: 1% acetic acid, 4% D.W./methanol, flow rate: 1 ml/min, column oven temperature: 35 ◦ C, detection: UV-Vis at 436 nm). 2.2.2. Evaluation of hypersensitivity reaction All test substances were powdery and unsuitable for the standard guinea pig maximization test because of their poor solubility or suspension properties in water or Freund’s complete adjuvant (FCA), with consequent difficulties in the intradermal injection procedure. Therefore, we used a modified guinea pig testing technique, the adjuvant and patch test, which has equivalent ability to detect hypersensitivity to the guinea pig maximization test (Sato et al., 1981), and is one of the recommended test methods in the guidelines for toxicity studies of drugs in Japan. 2.2.3. Adjuvant and patch test The test procedure followed the reported method (Sato et al., 1981). Briefly, aliquots of 0.1 ml each of a water-in-oil type emulsion (distilled water: FCA = 1:1) were injected intradermally into the four corners of a previously shaved shoulder region (2 cm × 4 cm). At the injection sites, scratches in the shape of a
S. Fujii / Toxicology 193 (2003) 261–267
263
Table 1 List of anthraquinone dyes examined Name
Structure
Quinizarin content Commercial grade (ppm)
D&C Green No. 5 (Alizarin Cyanin Green F, CI61570)
29
Purified grade (ppm)
1.2
440
<1
D&C Violet No. 2 (Alizurin Purple SS, CI60725)
4110
<1
Ext. D&C Violet No. 2 (Alizurol Purple, CI60730)
990
<1
D&C Green No. 6 (Quinizarin Green SS, CI61565)
.
grid were made with the needle used for injection. A closed patch with 0.1 ml of test preparation was applied to the sites for 24 h. Abrasions and sample applications were repeated on the following 2 days. One week after the initial sensitization, 10% sodium lauryl sulfate in petrolatum was applied to the intradermal injection sites. On the next day, a closed patch of the test preparation was applied at the same sites
Table 3 Evaluation of skin reaction (scale for scoring)
Table 2 List of hydroxyanthraquinones examined
Skin evaluation
Usual name
Chemical name
Quinizarin Alizarin Anthrarufin Chrysazin Purpurin Anthrapurpurin
1,4-Dihydroxyanthraquinone 1,2-Dihydroxyanthraquinone 1,5-Dihydroxyanthraquinone 1,8-Dihydroxyanthraquinone 1,2,4-Trihydroxyanthraquinone 1,2,7-Trihydroxyanthraquinone
Anthraquinone:
for 48 h. Two weeks later, a test sample was applied onto the shaved skin of the back. The excess substance was removed from the skin by washing with acetone at 24 h after the challenge application procedure. The skin reactions, fractional response (FR) and mean response (MR), were scored on the basis shown in Table 3 at 1, 24 and 48 h after the washing.
.
Scale for scoring
1. Erythema formation No erythema Very slight erythema Well-defined erythema Moderate to severe erythema Severe erythema with slight eschar formation
0 1 2 3 4
2. Edema formation No edema Slight edema Moderate edema Severe edema
0 1 2 3
Overall maximum score Mean response = n1 [(1) + (2)]/total number of animals.
7
264
S. Fujii / Toxicology 193 (2003) 261–267
Table 4 Contact hypersensitivity of D&C Green No. 6 Challenge substance
D&C Green No. 6 (commercial)
D&C Green No. 6 (purified) Quinizarin p-toluidine
Concentration (%)
1 0.1 0.01 0.001 1 0.1 0.01 1 1
Sensitized group
Control group
FR
MR
FR
MR
8/10 7/10 2/10 0/10 0/10 0/10 0/10 8/10 0/10
2.1 1.4 0.3 0 0 0 0 3.0 0
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
0 0 0 0 0 0 0 0 0
Induction: 1% D&C Green No. 6 (commercial grade). Solvent: acetone, FR: fractional response, MR: mean response.
3. Results The results obtained with commercial-grade and purified D&C Green No. 6 are summarized in Table 4. When guinea pigs were sensitized with 1% commercial-grade D&C Green No. 6, a positive dose-dependent response was obtained to challenge with the commercial-grade dye. However, no response was detected to 1% purified D&C Green No. 6. Quinizarin, a synthetic intermediate of the dye, gave a positive reaction at 1%, while p-toluidine, another intermediate, did not. Next, several commercial-grade and purified anthraquinone dyes were examined for contact hypersensitivity in guinea pigs sensitized with 0.2% quinizarin. As shown in Table 5, three commercial-grade dyes, D&C Green No. 6, D&C Violet No. 2 and Ext. Violet No. 2, gave positive reactions, though purified samples of these three dyes did not. In the case of D&C Green No. 5, neither commercial grade nor purified dye induced any reaction. Quinizarin gave a strong positive reaction even at 0.2%. The results of cross-reactivity testing of the four purified dyes in guinea pigs with 1% purified dyes are shown in Table 6. Purified D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 did not show any positive reaction, though purified D&C Violet No. 2 gave a positive reaction. The results of contact hypersensitivity testing of quinizarin are summarized in Fig. 1. Four different concentrations of quinizarin were used for the in-
Table 5 Cross-reactivity of anthraquinone dyes in guinea pigs sensitized with quinizarin Challenge substance
Concentration (%)
Sensitized group
Control group
FR
MR
FR
MR
D&C Green No. 5 Commercial 1 Purified 1
0/14 0/14
0 0
0/10 0/10
0 0
D&C Green No. 6 Commercial 1 Purified 1
5/14 0/14
1.1 0
0/10 0/10
0 0
D&C Violet No. 2 Commercial 1 Purified 1
8/14 0/14
1.1 0
0/10 0/10
0 0
Ext. D&C Violet No. 2 Commercial 1 Purified 1
1/14 0/14
0.1 0
0/10 0/10
0 0
Quinizarin
14/14
4.6
0/10
0
0.2
Induction: 0.2% quinizarin. Solvent: acetone, FR: fractional response, MR: mean response.
duction and challenge procedures in the experiments. Guinea pigs sensitized with 0.2% quinizarin did not give a positive reaction to challenge with 0.0002%, but positive reactions were confirmed at challenge
Fig. 1. Concentration dependence of contact hypersensitivity to quinizarin in guinea pigs. Four groups of guinea pigs (n = 10 for each group) were sensitized with four different concentrations of quinizarin and challenged with four different concentrations of quinizarin. Control group guinea pigs (n = 10) applied with four different concentrations of quinizarin were negative (FR = 0/10, MR = 0). Solvent: acetone.
S. Fujii / Toxicology 193 (2003) 261–267
265
Table 6 Cross-reactivity of anthraquinone dyes Challenge substance and concentration (%)
Induction substance D&C Green No. 5
D&C Green No. 6
D&C Violet No. 2
Ext. D&C Violet No. 2
FR
MR
FR
MR
FR
MR
FR
MR
Commercial 1 0.1 0.01 0.001
0/10 0/10 0/10 n.d.
0 0 0
0/10 0/10 0/10 n.d.
0 0 0
9/10 7/10 5/10 0/10
1.8 1.7 0.6 0
0/10 0/10 0/10 n.d.
0 0 0
Purified 1 0.1 0.01 0.001
0/10 0/10 0/10 n.d.
0 0 0
0/10 0/10 0/10 n.d.
0 0 0
10/10 10/10 8/10 0/10
2.2 2.0 0.9 0
0/10 0/10 0/10 n.d.
0 0 0
Quinizarin 0.2 0.02
0/10 0/10
0 0
0/10 0/10
0 0
3/10 0/10
0.3 0
0/10 0/10
0 0
Induction: 1% of each anthraquinone dye (purified grade). Solvent: acetone, n.d.: not done, FR: fractional response, MR: mean response. Control group: 1% of each dye and 0.2% of quinizarin were negative (FR = 0/10, MR = 0).
concentrations of 0.002, 0.02 and 0.2%. Guinea pigs induced with 0.0002% quinizarin did not show a positive reaction on challenge with 0.2%. Experiments on cross-reactivity among quinizarin and structurally-related substances were also performed. As shown in Table 7, quinizarin per se and purpurin per se showed positive reactions and these dyes showed cross-reactivity. Alizarin, anthrarufin, chrysazin and anthrapurpurin did not show either positive reaction or cross-reactivity among the evaluated substances.
4. Discussion The results of contact hypersensitivity testing of D&C Green No. 6 (Table 4) and cross-reactivity testing of purified anthraquinone dyes (Table 6) show that commercial-grade D&C Green No. 6 exhibited hypersensitivity reactions, whereas purified D&C Green No. 5, D&C Green No. 6 and Ext. D&C Violet No. 2 did not. The hypersensitivity to the commercialgrade dyes was concluded to be due to quinizarin, which is used in the synthesis of anthraquinone dyes
Table 7 Cross-reactivity of hydroxyanthraquinones Challenge substance
Induction substance Quinizarin
Quinizarin Alizarin Anthrarufin Chrysazin Purpurin Anthrapurpurin
Alizarin
Anthrarufin
Chrysazin
Purpurin
Anthrapurpurin
FR
MR
FR
MR
FR
MR
FR
MR
FR
MR
FR
MR
4/5 0/5 0/5 0/5 4/5 0/5
2.8 0 0 0 2.6 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
4/5 0/5 0/5 0/5 3/5 0/5
2.6 0 0 0 2.0 0
0/5 0/5 0/5 0/5 0/5 0/5
0 0 0 0 0 0
Induction: 0.5% each hydroxyanthraquinone in N-methylpyrrolidone. Challenge: 0.03% of each hydroxyanthraquinone in acetone, FR: fractional response, MR: mean response. Control group: 0.03% acetone solution of each substance was negative (FR = 0/5, MR = 0).
266
S. Fujii / Toxicology 193 (2003) 261–267
and is present as an impurity in commercial-grade dyes. In contrast, hypersensitivity to commercial-grade D&C Violet No. 2 appeared to be due to both the dye per se and quinizarin present as an impurity. D&C Violet No. 2 was reported not to be allergenic in the guinea pig maximization test with some modifications by Tomihata et al. (1999). They used a concentration of 5% in olive oil for intradermal injection, 10% in petrolatum for 48 h (closed application) at the induction stage and 10% in acetone for 24 h (open application) at challenge. In contrast, we used 1% in acetone for 24 h (closed application) for 3 consecutive days applied to water-in-oil emulsion (with FCA)-injected sites, 1% in acetone for 48 h (closed application) at the induction stage and maximum 1% in acetone for 24 h (closed application) at challenge. The differences between the reported results by Tomihata et al. (1999) and the results in Table 6 are likely to be due to the differences of experimental methods, especially at the challenge stage, and solvents used for the experiments. The adjuvant and patch test that we used was developed for the detection of weak allergens and to evaluate the allergenicity of materials unsuitable for testing by intradermal injection, and involves the use of FCA to stimulate the immune system of the animal and external application instead of intradermal injection of the test compound in the induction stage. It was reported that test results obtained by using the adjuvant and patch test correlated well with those obtained in the guinea pig maximization test (Sato et al., 1981). Interestingly, purified Ext. D&C Violet No. 2, a hydrophilic form of D&C Violet No. 2, did not induce hypersensitivity. A similar phenomenon was reported in relation to D&C Yellow No. 10 and D&C Yellow No. 11 (Sato et al., 1984). Strong and dose-dependent hypersensitivity to quinizarin was confirmed (Fig. 1). Guinea pigs sensitized with 0.002% quinizarin showed no reaction on challenge with 0.002%, though a weak positive reaction (FR = 1/10, MR = 0.1) was seen at a challenge concentration of 0.02%. These results indicate that guinea pigs could be sensitized with 0.002% quinizarin. Guinea pigs treated with 0.0002% quinizarin showed no positive reaction even to a challenge concentration of 0.2%. These results mean that guinea pigs were not sensitized by exposure to 0.0002% quinizarin. Quinizarin was reported to in-
duce allergic reactions when evaluated at 0.01% for intradermal injection, 10% for closed application in the induction stage and 0.01% for open application at the challenge stage. The results in Fig. 1 indicate stronger allergenicity of quinizarin than was previously reported (Tomihata et al., 2001), but the differences of test methods and solvents used in the experiments are likely to account for this. The reported results were obtained when quinizarin was evaluated at four different concentrations (0.0001–0.1%) in olive oil for intradermal injection, 10% in petrolatum for 48 h (closed application) at the induction stage and five different concentrations (0.0005–1.0%) in acetone for 24 h (open application) at challenge. In the present study, we evaluated the hypersensitivity to quinizarin by using the adjuvant and patch test, involving four different concentrations in acetone for 24 h (closed application) for 3 consecutive days on water-in-oil emulsion injected sites, four different concentrations in acetone for 48 h (closed application) at the induction stage and four different concentrations in acetone for 24 h (closed application) at challenge. The differences in test methods, especially at the challenge stage, and solvents used for the experiments might explain the stronger reaction to quinizarin in our study. In the experiment on cross-reactivity of hydroxyanthraquinones (Table 7), the results showed that 1- and 4-hydroxyl groups of anthraquinone-related compounds play a key role in hypersensitivity induction. It is important to note that anthraquinone-related substances, including some of the evaluated hydroxyanthraquinones, exist in plants. Generally, one group of these compounds has two or three hydroxyl groups on the benzene ring on one side of the molecule, while another group has one or two hydroxyl groups on benzene rings on both sides of the molecule. Examples include alizarin, purpurin, chrysophanol, emodin, aloe-emodin, rhein, etc. in madder, rhubarb and aloe (Okada and Mitsuhashi, 2002). It is known that sennoside, which is not an anthraquinone derivative, but an anthrone derivative, also exists in rhubarb, aloe, etc. A study on hypersensitivity induction by sennoside may be warranted. References Andrisano, V., Cavrini, V., Summer, P., Passuti, S., 1995. Determination of impurities in oxidation hair dyes as raw materials
S. Fujii / Toxicology 193 (2003) 261–267 by liquid chromatography (HPLC). Int. J. Cosmet. Sci. 17, 53–60. Brandao, F.M., Valente, A., 1988. Photodermatitis from anthraquinone. Contact Dermat. 18, 171–172. Buehler, E.V., 1965. Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol. 91, 171–177. Choquet-Kastylevsky, G., Descotes, J., 1998. Value of animal models for predicting hypersensitivity reactions to medicinal products. Toxicology 129, 27–35. Dooms-Goossen, A., Bonamte, A., Parys, M., Dooms, M., 1981. Sensitizing anthraquinone dye in spectacle frames. Contact Dermat. 7, 214–215. Hausen, B.M., Sawall, E.M., 1989. Sensitization experiments with textile dyes in guinea pigs. Contact Dermat. 20, 27–31. Kimber, I., 2001. The local lymph node assay and potential application to the identification of dug allergens. Toxicology 158, 59–64. Klecak, G., 1998. Test methods for allergic contact dermatitis in animals. In: Marzulli, F.N., Maibach, H.I. (Eds.), Dermatotoxicology Methods. Taylor & Francis, Bristol, PA, pp. 121–143. Kuo, K.L., Huang, H.Y., Hsieh, Y.Z., 1998. High-performance capillary electrophoretic analysis of synthetic food colorants. Chromatographia 47, 249–256. Magnusson, B., Kligman, A.M., 1969. The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52, 268–276. Maurer, T., 1996. Guinea pig predictive tests. In: Kimber, I., Maurer, T. (Eds.), Toxicology of Contact Hypersensitivity. Taylor & Francis, Bristol, PA, pp. 107–126. Miller, M.M., Goldgerg, H.S., Wilkerson, W.G., 1978. Allergic contact dermatitis to 1,4-bis(isopropylamino)anthraquinone. Arch. Dermatol. 114, 1793–1794. Morrow, D.M., Rapaport, M.J., Strick, R.A., 1980. Hypersensitivity to aloe. Arch. Dermatol. 116, 1064–1065. Okada, M., Mitsuhashi, H., 2002. Newly Revised Illustrated Medicinal Plants of the World. Hokuryukan Co., Tokyo, pp. 45, 499, 618 and 743. Salstad, D.M., Tepper, J.S., Doerfler, D.L., Qasim, M., Selegrade, M.K., 1994. Evaluation of azo and two anthraquinone dyes for allergic potential. Fundam. Appl. Toxicol. 4, 569–577. Sato, Y., Katsumura, Y., Ichikawa, H., Kobayashi, T., Kozuka, T., Morikawa, F., Ohta, S., 1981. A modified technique of
267
guinea pig testing to identify delayed hypersensitivity allergens. Contact Dermat. 7, 225–237. Sato, Y., Kutsuna, H., Kobayashi, T., Mitsui, T., 1984. D&C Yellow Nos. 10 and 11: chemical composition analysis and delayed contact hypersensitivity testing in the guinea pigs. Contact Dermat. 10, 30–38. Seidenari, S., Montovani, L., Manzini, B.M., Pignatti, M., 1997. Cross-sensitization between azo dyes and para-amino compound. Contact Dermat. 36, 91–96. Seidenari, S., Giusti, F., Massone, F., Mantovani, L., 2002. Sensitization to disperse dyes in a patch test population over a five-year period. Am. J. Contact Dermat. 13, 101–107. Sugai, T., Takahashi, Y., Takagi, T., 1997. Pigmented cosmetic dermatitis and coal tar dyes. Contact Dermat. 5, 249–256. Tomihata, K., Suzuki, M., Sato, H., 1999. Skin sensitization study of a new resorbable suture containing D&C Violet No. 2 on guinea pig maximization test. Jpn. J. Dermatoallergology 7, 101–108. Tomihata, K., Suzuki, M., Sato, H., Kitagawa, M., 2001. Sensitizer contained in heat-decomposed dye. J. Biomed. Mater. Res. 15, 531–539. Tsuji, S., Okada, M., Akamatsu, Y., Tonogai, Y., 2000. Studies on rejected Food Blue No. 1 (Brilliant Blue FCF) Aluminium Lake. Bull. Natl. Inst. Health Sci. 118, 131–134. Ulter, W., Geier, J., Lessmann, H., Hausen, B.M., 2001. Contact allergy to Disperse Blue 106 and Disperse Blue 124 in German and Austrian patients, 1995 to 1999. Contact Dermat. 44, 173– 177. Ulter, W., Lessmann, H., Geier, J., Becker, D., Fucks, T., Richter, G., 2002. The spectrum of allergic (cross-)sensitivity in clinical patch testing with ‘para amino’ compounds. Allergy 57, 319– 322. van Och, F.M.M., Vandebriel, R.J., Prinsen, M.K., De Jong, W.H., Slob, W., van Loveren, H., 2001. Comparison of dose–response of contact allergens using the guinea pig maximization test and the local lymph node assay. Toxicology 167, 207– 215. Vial, T., Descotes, J., 1994. Contact sensitization assays in guinea-pigs: are they predictive of the potential for systemic allergic reactions? Toxicology 93, 63–75. Wilkinson, S.M., Brittain, J., Beck, M.H., 1994. Allergic contact dermatitis from an anthraquinone derivatives in a chemical plant. Contact Dermat. 30, 241–242.