2022 Volume 47 Issue 12 Pages 503-506
The electron spin resonance (ESR)-based photosafety test (ESR-PT) is a non-animal prediction test for photosafety evaluations that can be used even for hydrophobic chemicals; the method is based on the detection of singlet oxygen generation using ESR spectroscopy and showing high accuracy for compounds with known photosafety information. During the process of extending the application data for ESR-PT, we found three false-negative chemicals: bithionol, fenticlor and cilnidipine. These chemicals did not show the characteristic triplet signal of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-hydroxy-TEMPO), which is used as a classifier for positive or negative chemicals; instead, bithionol and fenticlor induced an apparent single peak signal with a g-value of 2.0048, while cilnidipine produced a small, fragmented signal. Bithionol and fenticlor reportedly induce free radicals, and positive phototoxic or photoallergic evidence have been reported. Although the small, fragmented signal observed for cilnidipine was confirmed to be identical to that of a phenylnitroxy radical by the computer simulation, the significance of this chemical for photosafety considerations may be low because cilnidipine has quite a low incidence of phototoxic or photoallergic reactions in humans. Accordingly, the current ESR-PT protocol should be improved to detect free radical photoproducts generated from chemicals such as bithionol and fenticlor, thereby helping to reduce false negatives in ESR-PT.
Phototoxicity or photoallergic reactions are caused by exposure to ultraviolet (UV) or visible light irradiation after oral or dermal applications of phototoxicants or photoallergens. These reactions are sometimes caused by cosmetic and pharmaceutical products, so photosafety evaluations of chemicals used in consumer products is important to prevent phototoxic or photoallergic reactions caused by these products. Recently, we developed a new photosafety test method, the electron spin resonance (ESR)-based photosafety test (ESR-PT), which is applicable even to hydrophobic chemicals; the method is based on the detection of singlet oxygen generation using ESR spectroscopy, and it has a prediction accuracy of 100% for chemicals with known photosafety information (Hinoshita et al., 2021).
During the process of extending the application data for ESR-PT, we found three false-negative chemicals: bithionol, fenticlor and cilnidipine. Bithionol and fenticlor are known photoallergens, and positive photo-patch test results have been reported (Burry, 1967). These two chemicals also reportedly react photochemically with proteins via free radical reactions (Delahanty et al., 1989), and protein binding is known as one of the key events in the skin sensitization process proposed by the Organization for Economic Co-operation and Development (OECD, 2012). On the other hand, only a small number of photosensitivity reactions to cilnidipine have been reported in humans (Aoki et al., 2016).
This report describes the ESR spectroscopic properties of the three false-negative chemicals mentioned above and their significance for photosafety evaluations.
Bithionol, fenticlor and sulisobenzone were obtained from Tokyo Chemical Industry (Tokyo, Japan), and cilnidipine and its structurally similar chemicals, nimodipine and nifedipine, were also obtained from the same manufacturer (Fig. 1). 4-Hydroxy-2,2,6,6-tetramethyl-piperidine (4-hydroxy-TEMP) (> 98%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Quinine hydrochloride dihydrate (quinine HCl) and ethanol (analytical grade) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan).
Chemical structure of bithionol, fenticlor, cilnidipine, nimodipine and nifedipine.
The ESR spectrum measurement was performed under the same experimental conditions previously described (Hinoshita et al., 2021). Test chemicals were prepared in ethanol at concentrations of 0.1, 1, or 10 mmol/L with 100 mmol/L 4-hydroxy-TEMP; the test mixtures were irradiated using a xenon arc-lamp in the cavity of an ESR spectrometer (JES-TE200; JEOL, Tokyo, Japan), and the ESR spectra were recorded at room temperature before and after irradiation. The ESR measurements were conducted using the following parameters: microwave frequency, 9.42 GHz; microwave power, 4.0 mW; modulation width, 0.1 mT; magnetic field, 336 ± 5 mT; sweep width, 30 sec; time constant, 0.03 sec; amplitude, 600; and accumulation, 4. Then, the singlet oxygen formation (SOF) value was calculated using the following equation: SOF = (RIa–RIb) / (RIca–RIcb). To determine the relative intensity (RI), the peak height of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-hydroxy-TEMPO) relative to the MnO marker as an external standard, obtained from the test mixture before and after irradiation, was denoted as RIb and RIa, respectively. The relative intensity obtained from the control solution (ethanol containing 100 mmol/L 4-hydroxy-TEMP) before and after irradiation was denoted as RIcb and RIca, respectively. A tentative cutoff value (= 2.8) was used to discriminate positive/negative ESR-PT results.
To confirm the signal observed in cilnidipine and its significance to photosafety, the ESR spectra of cilnidipine and nimodipine at concentrations of 10 mmol/L without 4-hydroxy-TEMP were also measured in the same manner, nimodipine has no phototoxic or photoallergic evidence. In addition, to identify the ESR signal of cilnidipine, the ESR spectrum after the irradiation of this chemical was computer-simulated using an isotropic simulation program (IPRIT, JEOL, Tokyo, Japan), and the simulated ESR signal was compared with the observed ESR signals. ESR-PT was also conducted using nifedipine, a chemical that is structurally similar to cilnidipine and has multiple lines of evidence indicating phototoxicity (Seggev and Lagstein, 1996; Zenarola et al., 1991).
The ESR spectra of typical positive and negative chemicals, quinine HCl and sulisobenzone, after irradiation in the ESR-PT are shown in Fig. 2 (A) and Fig. 2 (B), respectively. Although both chemicals showed nearly no signals before irradiation, a characteristic triplet signal of 4-hydroxy-TEMPO generated by the reaction of singlet oxygen and 4-hydroxy-TEMP was observed at 336 mT only for quinine HCl after irradiation.
The electron spin resonance (ESR) spectra of (A) quinine HCl (10 mmol/L), (B) sulisobenzone (1 mmol/L), (C) bithionol (10 mmol/L), (D) fenticlor (10 mmol/L), (E) cilnidipine (10 mmol/L), (F) nimodipine (10 mmol/L) and (G) nifedipine (10 mmol/L) in ethanol in the presence of 4-hydroxy-TEMP (100 mmol/L) after irradiation. The symbols indicate following peaks: a lower field peak of 4-hydroxy-TEMPO resulting from the reaction of 4-hydroxy-TEMP and the singlet oxygen generated (closed diamond), a peak of the third signal of the MnO marker (open circle).
Positive evidence of phototoxicity or photoallergy has been reported for bithionol, fenticlor and cilnidipine (Burry, 1967; Aoki et al., 2016); however, these chemicals did not show the typical triplet signal of 4-hydroxy-TEMPO, as observed for quinine HCl, but rather showed a single peak or small, fragmented signals (Figs. 2 (C), (D), (E)). Consequently, these chemicals tested negative according to the current decision criteria of the ESR-PT, since the SOF values calculated for these chemicals were below the tentative positive criterion of a cutoff value of 2.8 (Table 1).
| Chemical name | CAS No. | Concentration (mmol/L)a | Mean SOF value | Judgment | Photosafety information |
|---|---|---|---|---|---|
| Positive/negative controls in ESR-PTb | |||||
| Quinine HCl | 6119-47-7 | 10 | 39.0 | + | + |
| Sulisobenzone | 4065-45-6 | 1 | 0.6 | – | – |
| Halogenated thiobisphenol | |||||
| Bithionol | 97-18-7 | 0.1 | 0.6 | – | +c |
| Fenticlor | 97-24-5 | 0.1 | 0.3 | – | +c |
| Calcium channel blocker | |||||
| Cilnidipine | 132203-70-4 | 0.1 | 0.3 | – | +d |
| Nimodipine | 66085-59-4 | 0.1 | 0.2 | – | ND |
| Nifedipine | 21829-25-4 | 10† | 8.1 | + | +e,f |
†, The chemical tested with the existence of precipitation.
Abbreviations: −, negative; +, positive; SOF, singlet oxygen formation; ND, no data.
aThe concentration given the highest SOF value in the preliminary tested from 0.1 mmol/L to 10 mmol/L.
bHinoshita et al. (2021), cBurry (1967), dAoki et al. (2016), eSeggev and Lagstein (1996), fZenarola and Lomuto (1991).
Bithionol and fenticlor induced an apparent single peak signal with a g-value of 2.0048 under the conditions of the ESR-PT (Figs. 2 (C), (D)). Although an origin of characteristic single peak signal with a g-value of 2.0048 could not be identified at present, bithionol and fenticlor are known to generate dehalogenated free radicals upon UV irradiation and to yield secondary free radical photoproducts, such as phenoxyl or semiquinone radicals (Li and Chignell, 1989). Accordingly, the observed signal was considered to have resulted from an interaction between 4-hydroxy-TEMP and dehalogenated free radicals or their secondary products generated from these chemicals, since almost no signal was detected without 4-hydroxy-TEMP (data not shown).
Nimodipine, which is structurally similar to cilnidipine and is used as a calcium channel blocker with no evidence of phototoxicity, produced a small, fragmented signal after irradiation under the current ESR-PT conditions, similar to that observed for cilnidipine (Fig. 2 (E), (F)). Meanwhile, nifedipine, which is also structurally similar to cilnidipine but has multiple lines of evidence indicating phototoxicity (Seggev and Lagstein, 1996; Zenarola et al., 1991), induced a typical triplet ESR signal and tested positive in the ESR-PT (Fig. 2 (G)).
To identify the signal generated from cilnidipine, the signals observed for cilnidipine and nimodipine in the absence of TEMP (Figs. 3 (A), (B)) were compared to the computer-simulated signal of nimodipine using IPRIT software (Fig. 3 (C)). Consequently, the signal observed for cilnidipine was confirmed to be identical to that of the phenylnitroxy radical induced by nimodipine after irradiation (g-value = 2.0057, aN = 0.93 mT, a1H(1) = 1.23 mT, a2H(3) = 0.30 mT, a3H(1) = 0.1 mT; Staško et al., 1994). These observations suggest that cilnidipine and nimodipine may generate a phenylnitroxy radical under the current ESR-PT conditions.
The electron spin resonance (ESR) spectra of (A) cilnidipine (10 mmol/L) and (B) nimodipine (10 mmol/L) in ethanol in the absence of 4-hydroxy-TEMP after irradiation and (C) computer-simulated spectrum of nimodipine.
Although cilnidipine and nimodipine were predicted to be positive by other non-animal tests for photosafety, such as a reactive oxygen species (ROS) assay and a micellar ROS assay based on superoxide generation (Onoue et al., 2008; Seto et al., 2013), these chemicals might be of minor importance for photoallergic or phototoxic responses because of the very low incidence of phototoxic or photoallergic reactions in humans. Meanwhile, nifedipine, which is structurally similar to cilnidipine but for which clear phototoxic evidence exists (Seggev and Lagstein, 1996; Zenarola et al., 1991), showed the typical triplet signal of 4-hydroxy-TEMPO (Fig. 2 (G)) and tested positive under the current ESR-PT conditions, since the SOF value was over 2.8 (Table 1).
In the present study, we examined the photochemical properties on the ESR-PT for bithionol, fenticlor and cilnidipine, which were recognized as false negatives according to the decision criteria used in a previous study (Hinoshita et al., 2021). Bithionol and fenticlor apparently have positive evidence of phototoxicity. However, cilnidipine has only a small number of photosensitivity reaction cases, and the incidence of phototoxic or photoallergic reactions in humans is quite low. Consequently, the significance of this chemical in terms of photosafety considerations may also be low. The current ESR-PT protocol was designed to detect only singlet oxygen formation in photosafety evaluations. Considering the above findings, the current ESR protocol should be improved to include the detection of free radical photoproducts, such as those generated from bithionol and fenticlor; such improvements would help to reduce false negatives in the ESR-PT.
Conflict of interestThe authors declare that there is no conflict of interest.
