Chem. Res. Toxicol. 2003, 16, 575-582
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Articles Structure Elucidation, Synthesis, and Contact Allergenic Activity of a Major Hydroperoxide Formed at Autoxidation of the Ethoxylated Surfactant C12E5 Anna Bodin,†,‡ Malin Linnerborg,‡ J. Lars G. Nilsson,† and Ann-Therese Karlberg*,†,‡ Department of Chemistry, Medicinal Chemistry, Dermatochemistry and Skin Allergy, Go¨ teborg University, SE-412 96 Go¨ teborg, Sweden, and Occupational Dermatology, National Institute for Working Life, SE-112 79 Stockholm, Sweden Received August 27, 2002
Ethoxylated alcohols, widely used as surfactants, are known to be susceptible to oxidation when exposed to air. At autoxidation, a complex mixture is formed, in which alkyl poly(ethylene glycol) aldehydes, alkyl poly(ethylene glycol) formates, hydroxyaldehydes, and formaldehyde have previously been identified. These compounds are all secondary oxidation products, some of which have been shown to be skin sensitizers and irritants. The primary oxidation products from ethoxylated alcohols are described in the literature as peroxides and hydroperoxides, but their structures have not been elucidated more closely. Peroxides and hydroperoxides are usually reactive species and can be suspected to be biologically active as skin sensitizers and irritants. In the present investigation, we studied the autoxidation of the pure ethoxylated alcohol pentaethylene glycol mono-n-dodecyl ether (C12E5), using NMR and HPLC-MS. On the basis of experience from previous studies on a small model compound, diethyleneglycol monoethyl ether (C2E2), the hydroperoxide expected to be found in the highest amount in autoxidized C12E5 was synthesized and used as a reference substance in the analyses. This same hydroperoxide, 16-hydroperoxy-3,6,9,12,15-pentaoxaheptacosan-1-ol, was identified in the autoxidation mixture of C12E5, and its sensitizing capacity was determined. It was found to be a moderate allergen in experimental sensitization studies in guinea pigs. Our data further indicate the presence of at least three additional hydroperoxides in the autoxidation mixture of C12E5, one of which was identified as 1-hydroperoxy-3,6,9,12,15-pentaoxaheptacosan-1-ol. The results accentuate the importance of controlling the storage, transportation, and handling conditions of ethoxylated surfactants, to avoid the formation of allergenic and skin irritant oxidation products.
Introduction Ethoxylated surfactants are widely used in household and industrial cleaners, in topical pharmaceuticals, cosmetics, and laundry products. The surface active agents function as emulsifiers as well as suspending, wetting, and solubilizing agents, which give the products physical stability (1). The surfactants also affect the permeability of the skin, which makes them suited as penetration enhancers in transdermal drug delivery systems (2). Nonionic surfactants are often preferred to ionic surfactants in topical products, since they are considered to cause less skin irritation (3). However, previous work from our group shows that autoxidation of nonionic alcohol ethoxylates generates products that are both skin irritants (4) and sensitizers (5, 6). * To whom correspondence should be addressed. Tel: +46 31 772 4726. Fax: +46 31 772 3840. E-mail:
[email protected]. † Go ¨ teborg University. ‡ National Institute for Working Life.
It is well-known that polyethers, e.g., ethoxylated surfactants and poly(ethylene glycol)s, are easily oxidized by atmospheric oxygen when stored and handled (7). We have shown that a chemically well-defined ethoxylated alcohol, pentaethylene glycol mono-n-dodecyl ether (referred to as C12E5), forms a complex mixture of autoxidation products when exposed to air. The autoxidation products identified until now are all secondary oxidation products (Figure 1) (5, 6, 8). However, in previous studies from our group, it has been shown that primary autoxidation products, e.g., hydroperoxides, are main sensitizers in both colophony and limonene (9, 10), which inspired us to investigate the primary autoxidation products from the ethoxylated surfactants. Recently, we identified the first primary autoxidation product from the small model compound diethylene glycol monoethyl ether (referred to as C2E2) (11). From a chemical point of view, this hydroperoxide, 2-[2-(1-hydroperoxyethoxy)ethoxy]ethanol (Figure 2), is likely to be the most stable hydroperoxide out of the six possible ones to be formed
10.1021/tx025609n CCC: $25.00 © 2003 American Chemical Society Published on Web 04/12/2003
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Chem. Res. Toxicol., Vol. 16, No. 5, 2003
Figure 1. Identified secondary autoxidation products from C12E5. Compound 5 was used in the synthesis of hydroperoxide 4.
Figure 2. Chemical structure of 2-[2-(1-hydroperoxyethoxy)ethoxy]ethanol.
at autoxidation of C2E2. The aim of the present study was to identify and determine the sensitizing capacity of one of the major hydroperoxides expected to be formed upon autoxidation of C12E5, according to our findings from studies of the model compound C2E2.
Experimental Procedures Caution: Skin contact with hydroperoxides must be avoided. As skin-sensitizing substances, these compounds must be handled with care. Chemicals. 3,6,9,12,15-Pentaoxaheptacosan-1-ol (pentaethylene glycol mono-n-dodecyl ether, C12E5, 98%) was purchased from Nikkol Chemicals Co., Ltd. (Tokyo, Japan) (C12E5). HPLCMS analyses of C12E5 dissolved in water/acetonitrile (1:3, v/v) yielded the sodium adduct of C12E5 of high intensity (m/z 429). The ammonium adduct (m/z 424) and the (M + 1)+ adduct (m/z 407) were also present in high intensity. NMR spectral characteristics of C12E5: 1H NMR (CDCl3): δ 0.86 (t, 3H, CH3), 1.241.36 (m, 20H, (CH2)10), 1.56 (m, 2H, CH2CH2CH2O), 3.43-3.71 (m, 20H, (CH2O)10). 13C NMR (CDCl3): δ 14.1 (CH3), 22.6 (CH2CH3), 22.6-29.6 ((CH2)10), 61.7 (CH2CH2OH), 70.3-71.8 ((CH2O)11). Dicyclohexylcarbodiimide (DCC, 99%), dodecanal (95%), dodecanoic acid (98%), hydrogen peroxide (35%), (methoxymethyl)triphenylphosphonium chloride (98%), and pentaethylene glycol (98%) were purchased from Lancaster (Lancashire, U.K.). 4-(Dimethylamino)pyridine (99%) was obtained from Kebo Lab
Bodin et al. AB (Spånga, Sweden). Lithium diisopropylamide (LDA), 1.5 M solution in THF, was purchased from Aldrich (Milwaukee, WI). Instrumentation and Mode of Analysis. NMR spectroscopy was performed on a JEOL Eclipse 400 instrument, using CDCl3 solutions. Chemical shifts (ppm) are reported relative to tetramethylsilane (TMS) (δH 0.00) as reference. HPLC-MS analyses were performed using electrospray ionization (ES) on a Hewlett-Packard 1100 HPLC-MS. The HPLC was equipped with a Zorbax SB-C18 column (150 mm × 3.0 mm i.d.) with a particle size of 3.5 µm (Chromtech, Stockholm, Sweden). Water/ acetonitrile (1:3, v/v) was used as eluent. Formic acid (5 mM) was added to promote the formation of positive ions. The flow rate was 0.50 mL/min, and the column temperature was set to 25 °C. The mass spectrometer was equipped with an ES interface run in the positive ionization mode with nebulizer pressure, 30 psi; capillary voltage, 4000 V; drying gas temperature, 350 °C; and drying gas flow rate, 10 L/min. Fragmentor voltage was set to 60 V. The scan mode was chosen for identification of hydroperoxides and ester in oxidized C12E5 and for identification of synthesized material. Selected ion monitoring was chosen for determination of degree of oxidation of C12E5, and m/z values were monitored as follows: 407.4 (protonated molecular ion of C12E5) and 345.2 (qualifier fragment of C12E5). GC-MS was performed on a Hewlett-Packard model 6890 instrument equipped with a cool on-column capillary inlet and an HP-5MS fused silica capillary column (30 m × 0.25 mm, 0.25 µm, Hewlett-Packard, Palo Alto, CA). The temperature program was beginning at 70 °C, increasing with 20 °C/min, and finishing at 270 °C for 5 min. Helium was used as the carrier gas, and ionization was achieved using electron ionization at 70 eV. Autoxidation of C12E5. Two different samples of neat C12E5 were stored in open Erlenmeyer flasks in daylight at room temperature during 16 and 26 months, respectively. The tops of the flasks were covered with aluminum foil to prevent contamination and to diminish evaporation. The samples were stirred for 1 h four times daily to mimic regular handling. The degree of oxidation after 16 and 26 months, respectively, measured as percent remaining starting material, was determined by HPLC-MS. Synthesis. 1. 1-Methoxytridecene (1). Compound 1 was synthesized from laurylaldehyde according to a method described by Yamazaki et al (12). A solution of LDA in THF (1.5M) (10 mL, 13.5 mmol) was added to a suspension of (methoxymethyl)triphenylphosphonium chloride (5.00 g, 14.6 mmol) in toluene (30 mL) at 0 °C under nitrogen atmosphere, and the mixture was stirred for 10 min. Laurylaldehyde (2.49 g, 13.5 mmol) in toluene (30 mL) was added, and the reaction mixture was stirred for 50 min. The reaction was quenched by addition of saturated aqueous NH4Cl. The water phase was extracted with ether (3 × 50 mL), and the combined organic phases were washed with saturated NaCl (3 × 50 mL), dried with MgSO4, and concentrated. The residue was purified by silica gel column chromatography (toluene-pentane 1:2) affording (1.3 g) 45% of pure 1. The GC chromatogram of the synthesized 1 contained two peaks, in a 66:34 ratio, with identical mass spectra showing the characteristic molecular ion at m/z 212 and m/z 180 after loss of methanol, identified as the E and Z isomers. NMR spectral characteristics, E/Z mixture: 1H NMR (CDCl3): δ 0.87 (t, 3H, CH3), 1.15-1.35 (m, 18H, (CH2)9), 1.89/2.03 (m, 2H, CH2CHdCH),), 3.49/3.56 (s, 3H, OCH3), 4.33/4.72 (m, 1H, CHdCHO), 6.27/5.85 (d, 1H, CHdCHO. 13C NMR (CDCl3): δ 14.2 (CH3), 22.8-32.0 ((CH2)10), 55.9/59.5 (CH2CHdCHO), 103.3/ 107.3 (CHdCHO), 146.9/146.0 (CH)CHO). 2. 16-Hydroperoxy-3,6,9,12,15-pentaoxaheptacosan-1-ol (2). The transformation of enol ether 1 to hydroperoxyketal 2 by ozonolysis was performed according to a method described by Dussault et al. (13). A solution of 1 (1.3 g, 6.1 mmol) and pentaethylene glycol (2.2 g, 9.4 mmol) in dichloromethane (25 mL) was cooled to -70 °C. Ozone was passed through the solution for 10 min. Excess ozone was removed with a stream of oxygen. The solvent was evaporated under reduced pressure at ambient temperature. The resulting crude product was
Autoxidation of Ethoxylated Alcohols dissolved in dichloromethane (5 mL) and washed with water (5 × 2 mL). The organic phase was dried (MgSO4) and concentrated, affording (1.1 g) 41% of pure hydroperoxyketal 2. HPLC-MS analyses of synthesized 2 dissolved in water/ acetonitrile (1:3, v/v) yielded the ammonium adduct (m/z 456) and the sodium adduct (m/z 461) of 2 of high intensity. Also, (M - OOH)+ was present at m/z 405, as well as the potassium adduct at m/z 477. NMR spectral characteristics: 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.23-1.35 (m, 18H, (CH2)9), 1.61 (m, 1H, CH2CH2CHOOH),), 1.70 (m, 1H, CH2CH2CHOOH), 3.62-3.74 (m, 18H, (CH2O)9), 3.85 (t, 2H, CH2OCHOOH), 4.85 (t, 1H, CHOOH). 13C NMR (CDCl3): δ 14.1 (CH3), 22.67 (CH2CH3), 24.9 (CH2CH2CHOOH), 29.3-29.6 ((CH2)6), 31.2 (CH2COOH) 31.9 (CH2CH2CH3) 61.74 (CH2OH), 61.67 (CH2OH), 65.1 (CH2OCHOOH), 70.2-70.6 ((CH2O)7), 72.5 (OCH2CH2OH), 107.1 (CHOOH). Synthetic 2 was degraded to 3 when stored at room temperature. The degradation product had identical chromatographic and spectral properties as the synthetic compound 3 described below. 3. 14-Hydroxy-3,6,9,12-tetraoxatetradecyl Laurate (3). The synthesis of 3 was performed according to a method described by Lopez et al. (14). A solution of DCC (1.33 g, 6.5 mmol) and 4-(dimethylamino)pyridine (DMAP) (0.06 g, 0.5 mmol) in dry acetonitrile (15 mL) was mixed with dodecanoic acid (1.00 g, 5 mmol) under nitrogen atmosphere. Pentaethylene glycol (1.78 g, 7.5 mmol) was added, and the mixture was stirred at room temperature for 1 h. The solution was then refluxed overnight, cooled, filtered, and extracted with ethyl ether (3 × 50 mL). The organic phase was dried (MgSO4) and evaporated under reduced pressure. The product was purified on a silica gel column eluted with ethyl acetate containing 0-10% methanol. Pure 3 was obtained in (1.47 g) 70% yield. HPLC-MS analyses of the pure, synthesized 3 dissolved in water/acetonitrile (1:3, v/v) yielded the ammonium adduct (m/z 438) and the sodium adduct (m/z 443) of 3 of high intensity. Also, (M + 1)+ was present at m/z 421. NMR spectral characteristics: 1H NMR (CDCl3): δ 0.88 (t, 3H, CH3), 1.23-1.35 (m, 14H, (CH2)7), 1.62 (p, 2H, CH2CH2CH2CO), 2.33 (t, 2H, CH2CH2CO), 3.61-3.74 (m, 18H, (CH2O)9), 4.23 (t, 2H, CH2OCO). 13C NMR (CDCl3): δ 14.1 (CH3), 22.7 (CH2CH3), 24.9 (CH2CH2CO), 29.2-31.9 ((CH2)8), 34.2 (CH2CO), 61.7 (CH2CH2OH), 63.4 (CH2OCO) 69.2-70.6 ((CH2O)7), 72.5 (OCH2CH2OH), 173.9 (CO). 4. 1-Hydroperoxy-3,6,9,12,15-pentaoxaheptacosan-1-ol (4). Dodecyltetraoxyethyleneoxyacetaldehyde (5) (Figure 1b) was synthesized as previously described (5). Compound 5 (1.05 g, 2.6 mmol) was mixed with hydrogen peroxide (0.60 g, 13.1 mmol), and the mixture was stirred at room temperature overnight. The product was too unstable to purify, which was why no yield could be calculated. HPLC-MS analyses of the reaction mixture containing synthesized 4 dissolved in water/ acetonitrile (1:3, v/v) yielded the ammonium adduct (m/z 456) and the sodium adduct (m/z 461) of 4 of high intensity. Also, (M - OOH)+ was present at m/z 405, as well as the potassium adduct at m/z 477. Studies on the Sensitizing Capacity of 2 and 3. The sensitizing capacity of 2 and 3 was determined using female Dunkin-Hartley guinea pigs (weight 300-400 g) from HB Lidko¨pings kaninfarm (Lidko¨ping, Sweden). The animals were kept on a standard diet from Beekey (North Humberside, England) and water ad libitum. They were randomly assigned to two exposed groups, designated A (n ) 16) and B (n ) 16), and one control group (n ) 16). The sensitization study was performed according to a modified protocol (15) of the cumulative contact enhancement test (CCET) (16), using closed challenge testing and sham-treated controls. All animals received an occlusive epidermal application on their shaved upper back on days 0, 2, 7, and 9 as induction. Group A was induced with 2 (114 mM, 5% w/w) in distilled water, group B was induced with 3 (643 mM, 27% w/w) in distilled water, and the control group was induced with just distilled water. On each of the four induction occasions, a filter paper (4 × 2 cm), wetted with about 200 mg of the induction material, was attached to the skin. All
Chem. Res. Toxicol., Vol. 16, No. 5, 2003 577 Table 1. Sensitization Studies on 2 in Guinea Pigs Using the CCET Method in a Modified Versiona no. of animals with positive reactions first challenge (day 22) 0.3% 48 h 72 h
4 10c
48 h 72 h
1 0
0.1%
water
exposed group 0 0 1 0 0 0
rechallenge (day 65) 0.4% Ab
0.3%
(n ) 16) 6d 4e 5e 5e
control group (n ) 16) 0 0 0 0
0 0
0.2%
water
3 6e
0 0
1 1
0 0
a Results from first challenge and rechallenge. b Induction: 5.0% w/w (114 mM) 2 in distilled water. c Significantly different from controls, p < 0.001 (n reactions in the exposed group as compared to n reactions in the control group). d Significantly different from controls, p < 0.01. e Significantly different from controls, p < 0.05.
animals were injected with Freund’s complete adjuvant (FCA, Difco, Detroit, MI) (0.1 mL × 2), intradermally on the upper back, on day 7. Challenge testing on the shaved flanks was performed on day 22 using small Finn Chambers (8 mm i.d., Epitest, Helsinki, Finland). Group A was tested with 2 in the concentrations 2.28 and 6.85 mM (0.1 and 0.3% w/w) in distilled water and with a vehicle control (Table 1). Group B was tested with 3 in the concentrations 71.4, 214, and 643 mM (3.0, 9.0 and 27% w/w) in distilled water and with a vehicle control. The control group was tested with all test solutions and the vehicle control. A test solution of 15 µg was applied in each Finn Chamber. After 24 h, the chambers were removed. Visual evaluation of the reactions was performed at 48 and 72 h after test application. The minimum criterion for a positive reaction was a confluent erythema. Rechallenge was performed on group A and the control group on day 65. These animals were tested with 2 in the concentrations 4.57, 6.85, and 9.13 mM (0.2, 0.3, and 0.4% w/w) in distilled water (Table 1). The animals from group A received an epidermal booster dose of 2 (114 mM in distilled water) 15 days before the retesting in order to stimulate the specific T-lymphocytes that were developed in the sensitization phase. Prior to the experiments, a test was performed in FCA-treated animals, to obtain the lowest irritating and the highest nonirritating concentrations of 2 and 3 for challenge testing. The study was approved by the local ethics committee. Statistical Analyses. The results from the sensitization study were statistically evaluated using Fisher’s exact test. The number of reactions in the exposed group was compared to the number of reactions in the control group. A p value of
