Elsevier

Toxicology in Vitro

Volume 28, Issue 4, June 2014, Pages 515-523
Toxicology in Vitro

Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators

https://doi.org/10.1016/j.tiv.2013.11.014Get rights and content

Highlights

  • ROS assay was developed for the photosafety assessment of pharmaceutical substance.

  • Present study aimed to validate a protocol for ROS assay using two different solar simulators.

  • Although several false positives appeared, there were no false negative predictions in both solar simulators.

  • Validation study demonstrated satisfactory transferability, accuracy, precision, and predictivity.

Abstract

A previous multi-center validation study demonstrated high transferability and reliability of reactive oxygen species (ROS) assay for photosafety evaluation. The present validation study was undertaken to verify further the applicability of different solar simulators and assay performance. In 7 participating laboratories, 2 standards and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals, were assessed by the ROS assay using two different solar simulators (Atlas Suntest CPS series, 3 labs; and Seric SXL-2500V2, 4 labs). Irradiation conditions could be optimized using quinine and sulisobenzone as positive and negative standards to offer consistent assay outcomes. In both solar simulators, the intra- and inter-day precisions (coefficient of variation; CV) for quinine were found to be below 10%. The inter-laboratory CV for quinine averaged 15.4% (Atlas Suntest CPS) and 13.2% (Seric SXL-2500V2) for singlet oxygen and 17.0% (Atlas Suntest CPS) and 7.1% (Seric SXL-2500V2) for superoxide, suggesting high inter-laboratory reproducibility even though different solar simulators were employed for the ROS assay. In the ROS assay on 42 coded chemicals, some chemicals (ca. 19–29%) were unevaluable because of limited solubility and spectral interference. Although several false positives appeared with positive predictivity of ca. 76–92% (Atlas Suntest CPS) and ca. 75–84% (Seric SXL-2500V2), there were no false negative predictions in both solar simulators. A multi-center validation study on the ROS assay demonstrated satisfactory transferability, accuracy, precision, and predictivity, as well as the availability of other solar simulators.

Introduction

Drug-induced phototoxicity can appear in light-exposed tissues, elicited by topical or systemic application of drugs and exposure to sunlight or artificial light (Moore, 2002). Several classes of pharmaceutics cause phototoxic reactions in skin and/or eyes (Moore, 1998, Moore, 2002), including photoirritant, photoallergic, and photogenotoxic events (Epstein, 1983). Although drug-induced phototoxicity might not be a life-threatening side effect in most cases, phototoxicity has a major impact on quality of life and therapeutic compliance/outcomes. With the aim of reducing and preventing phototoxicity, increasing attention has been drawn to hazard identification and risk management upon photosafety assessment of pharmaceutical products. A number of in vitro methodologies have been developed for photosafety assessment over the past few years. Guidance on the photosafety testing of medicinal products was established by regulatory agencies in the US and EU in the early 2000s (Seto et al., 2012), and the recent issuance of the draft ICH S10 photosafety guidance document also provided a detailed framework and guidance for photosafety evaluation of pharmaceutical substances and products (ICH, 2013). These guidelines describe photosafety assessment strategies on the basis of photochemical and photobiochemical properties, and in vivo pharmacokinetic behavior (EMEA/CPMP, 2002, FDA/CDER, 2002, OECD, 2004).

Previously, a reactive oxygen species (ROS) assay was developed for the photosafety assessment of pharmaceutical substances (Onoue et al., 2008b, Onoue and Tsuda, 2006), in which the generation of ROS such as singlet oxygen and superoxide from photoirradiated chemicals was monitored. The photo-excited phototoxins tend to generate ROS, triggering phototoxic events in the light-exposed tissues (Brendler-Schwaab et al., 2004, Epstein and Wintroub, 1985), and the photobiochemical responses of phototoxins could provide a rationale for the use of ROS assay in photosafety assessment. A multi-center validation study was previously carried out to establish and validate a standard protocol for the ROS assay, supervised by the Japanese Center for the Validation of Alternative Methods (JaCVAM) (Onoue et al., 2013). Outcomes from the validation study were indicative of the satisfactory transferability, inter-laboratory variability, and predictivity of the ROS assay, and these findings provided sufficient support for the ROS assay as an alternative method for photosafety assessment. However, the ROS assay in the previous validation study was conducted in only one solar simulator (Atlas Suntest CPS series), so the applicability of other solar simulators to the ROS assay has never been elucidated.

The present study was designed to validate a standard protocol for the ROS assay using different solar simulators, under the supervision of the Japanese Center for the Validation of Alternative Methods (JaCVAM) throughout the work. Since a UVA light source has been widely employed for the 3T3 neutral red uptake phototoxicity test (3T3 NRU PT) (Spielmann et al., 1994b), the present validation study focused on the compatibility of another solar simulator (Seric SXL-2500V2) commonly used for 3T3 NRU PT as an alternative to the Atlas Suntest series. In accordance with the previous study design, inter- and intra-laboratory validation studies were carried out to assess the transferability, assay precision, and predictive capacity of the ROS assay using 2 standard chemicals and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals.

Section snippets

General conditions of the study

The validation study was coordinated as reported previously (Onoue et al., 2013). Briefly, the Validation Management Team (VMT) was organized under the JaCVAM, and the roles of the VMT were to design the study, to guide and facilitate the validation process, to evaluate the results and, on the basis of these, render subsequent decisions during the progress of the study, and to analyze the outcomes from the studies. The VMT was comprised of the trial coordinator, assistant trial coordinator,

Optimization of irradiance conditions for transferable assay

A previous validation study demonstrated the satisfactory accuracy, precision, and prediction capacity of ROS assay using the Atlas Suntest CPS series at an irradiance intensity of ca. 2.0 mW/m2 (Onoue et al., 2013). According to the results from preliminary studies with a focus on irradiance conditions, ROS assay data on quinine (1), a typical phototoxin (Moore, 2002), and sulisobenzone (2), a non-phototoxic chemical (Portes et al., 2002), using the Seric SXL-2500V2 (Lab#4–7) at an intensity of

Conclusion

In the present validation study, the photochemical reactivities of 42 coded chemicals and 2 standard controls were assessed by the ROS assay using Seric SXL-2500V2, and the outcomes were compared with the previous validation data using the Atlas Suntest CPS series. The ROS assay using two different solar simulators achieved satisfactory transferability of the method, fine intra-/inter-laboratory reproducibility, and predictive capacity. On the basis of the present findings, upon spectral

Conflict of interest

None of the authors have any conflicts of interest associated with this study.

Acknowledgements

This work was supported in part by a Health Labour Sciences Research Grant from The Ministry of Health, Labour and Welfare, Japan. The authors are grateful to Dr. Manfred Liebsch (ZEBET), ICCVAM, ECVAM, and KoCVAM for valuable suggestions throughout this work.

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