False-negative chemicals in ESR-based photosafety test (ESR-PT) and their significance for photosafety evaluations: examples of bithionol, fenticlor and cilnidipine

Abstract

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.

Modified ESR‐based photosafety test (ESR‐PT) detecting singlet oxygen and free radical formation

“…Fifty‐two chemicals out of 56 chemicals produced the typical triplet signal of 4‐hydroxy‐TEMPO with a g ‐value of 2.006 and a hyperfine coupling constant of 1.60 mT, resulting from the reaction between 4‐hydroxy‐TEMP and singlet oxygen generated from the chemicals (Figure 2A) or background signal as observed in the negative control chemical (Figure 2B). Two chemicals, bithionol and fenticlor (10 mmol/L), produced single peak signals with a g ‐value of 2.005 (Figure 2C), whereas cilnidipine (10 mmol/L) produced a small, fragmented signal with a g ‐value of 2.006 (Figure 2E), as previously mentioned (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2022). Besides, doxycycline HCl (10 mmol/L) produced signals that were split into more than four peaks (Figure 2D).…”

“…The original ESR‐PT based on the detection of singlet oxygen formation is a promising non‐animal test method for photosafety evaluation of chemicals in terms of their applicability to hydrophobic chemicals (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2021). However, in previous research, we have indicated a need to improve the original ESR‐PT to prevent false negative results from chemicals that generate radical species other than singlet oxygen (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2022). To improve the ESR‐PT to include the detection of free radical photoproducts generated from test chemicals, PRI was newly used as a parameter to determine positive or negative test results.…”

“…In the original ESR‐PT, singlet oxygen formation (SOF) is used as a classifier for determining positive/negative results of the photosafety properties of chemicals. However, in previous research, we have suggested that certain chemicals produce free radicals and generate false negative results in the original ESR‐PT and further indicated that detection of both singlet oxygen and chemical‐derived free radicals would reduce the rate of false negative results by ESR‐based photosafety tests (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2022).…”

“…Cilnidipine has had few cases of photoallergic reaction, and the incidence of phototoxic or photoallergic reactions in humans is quite low. Moreover, the significance of this chemical in terms of photosafety considerations may be low (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2022). Therefore, the criteria for modified ESR-PT using new parameters, PRI and cutoff value of 2.0, have been confirmed to be effective in distinguishing chemicals with phototoxic and/or photoallergic reaction concerns, regardless of the photoproduced species.…”

“…During the process of extending the application data to additional chemicals for ESR‐PT, we found false negative chemicals showing a single peak signal instead of the characteristic triplet signal, and the single peak signal was considered to be derived from free radical photoproducts generated from chemicals, suggesting that an addition of the detection of these free radicals to the original ESR‐PT protocol could lead to the expansion of the applicability domain of ESR‐PT and contribute to the reduction in false negative results (Hinoshita, Abe, Sato, Maeda, & Takeyoshi, 2022). In this study, we aimed to establish a modified ESR‐PT using a new parameter for simultaneous detection of singlet oxygen and free radical photoproducts generated by chemicals.…”