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

The electron spin resonance-based photosafety test (ESR-PT) was modified using a new parameter, photoreactivity index (PRI), to detect singlet oxygen and free radical photoproducts simultaneously. With this modification, the modified ESR-PT is expected to reduce the number of false negative results due to chemicals producing free radical photoproducts other than singlet oxygen. The assay performance of the modified ESR-PT was evaluated using 56 chemicals, including hydrophobic chemicals. When using the PRI cutoff value of 2.0 in the modified ESR-PT, the accuracy relative to photosafety reference data was 91.1%, and the applicability (100%) was better than the other non-animal photosafety test. Among the chemicals producing positive results, bithionol, fenticlor, and doxycycline HCl were considered positive based on the detection of free radical photoproducts, suggesting that these three chemicals may have phototoxic or photoallergic potential via radical reactions. Additionally, this finding demonstrated the fundamental advantage of the modified ESR-PT using ESR spectroscopy, which can detect radicals selectively and quantitatively. Accordingly, the new parameter PRI is effective for photosafety evaluations based on not only singlet oxygen but also free radical photoproducts generated from chemicals. Therefore, the modified ESR-PT has a great potential for a photosafety test method applicable to various chemicals.

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

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.

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance.

Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals. In the present study, we attempted to develop a new photosafety test method, named the electron spin resonance-based photosafety test (ESR-PT), that would be applicable even to hydrophobic chemicals based on the detection of singlet oxygen generation after irradiation using ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine as a spin trap reagent. To achieve a quantitative evaluation, the singlet oxygen formation (SOF) value, which can be calculated as the increment in relative intensity after irradiation of the test mixture normalized by the increment in relative intensity after irradiation of the vehicle control solution, was calculated. The performance of the ESR-PT was evaluated by testing all the proficiency chemicals of the ROS assay plus additional chemicals, including hydrophobic chemicals and chemicals that tested false negative in the 3T3-NRU PT and ROS assay. SOF values were successfully calculated for all the chemicals tested including the hydrophobic chemicals, and the accuracy of the ESR-PT using a tentative cutoff value of 2.8 against the photosafety information was 100%. Therefore, the SOF value could be an effective parameter for photosafety evaluations, suggesting that the newly developed ESR-PT is a promising non-animal test applicable even to hydrophobic chemicals.

Low-magnetic field effect and electrically detected magnetic resonance measurements of photocurrent in vacuum vapor deposition films of weak charge-transfer pyrene/dimethylpyromellitdiimide (Py/DMPI) complex.

Magnetic field effect (MFE) and electrically detected magnetic resonance (EDMR) measurements of photocurrent have been conducted to clarify the excited-state dynamics in films of an organic weak charge-transfer (CT) complex, Pyrene/Dimethylpyromellitdiimide (Py/DMPI), fabricated by vacuum vapor deposition. Low-field MFE measurements of the photocurrent were carried out using an interdigitated platinum electrode made on a quartz substrate as well as the re-examination of the photocurrent and MFE in the range of 3-200 mT. The spin-dependent carrier dynamics leading to the low-field MFE are reasonably simulated as the low-field effect due to the hyperfine mechanism in the radical-pair intersystem crossing, which was solved through the Liouville equations of the density matrix for the stepwise hopping model in the doublet electron-hole pair (DD pair mechanism). Single-crystal time-resolved electron spin resonance measurement was also carried out to justify the MFE mechanism. The averaged trap depth (Etrap) of the triplet exciton was estimated to be +640 ± 89 cm-1 (Etrap/kB = +921 ± 128 K) by the temperature dependence of the signal intensity. This finding gave confidential experimental evidence for the majority of the trapped triplet exciton (3ext). The EDMR experiment directly revealed the evidence of the weakly coupled electron-hole pairs. The effective activation energies (ΔE) for the separation from the photoinduced CT state to the mobile carries are 1200-1900 cm-1 (ΔE/kB = 1700-2700 K). A systematic protocol to clarify the photo-generated carrier dynamics in weak CT complexes is demonstrated, and our findings from this method give not only further support for the two types of collision mechanisms assumed in our previous work but also the detailed information of the carrier dynamics of the weak CT complex, including the activation energy and trapping/detrapping process, which have significant influence on the performance of the organic devices.

Photoconductivity and magnetoconductance effects on vacuum vapor deposition films of weak charge-transfer complexes.

Thin films of weak charge-transfer (CT) complexes (pyrene/dimethylpyromellitdiimide or pyrene/pyromellitic dianhydride) were prepared on an interdigitated platinum electrode by vacuum vapor deposition. Their photoconductivity and magnetoconductance (MC) effects were investigated, and mobile triplet excitons (probably CT excitons) were detected by time-resolved ESR (TRESR) at room temperature. The MC effect on the photocurrent was observed and analyzed by quantum-mechanical simulation assuming two types of collision mechanisms between the electron and hole carriers and between the trapped triplet excitons and mobile carriers. A successful simulation was achieved when the parameters (g, D, E, and polarization) determined by TRESR and the effective hyperfine splitting estimated from an ab initio molecular-orbital calculation were used.