The in vivo rat skin photomicronucleus assay: phototoxicity and photogenotoxicity evaluation of six fluoroquinolones

Astrid A. Reus 2 Mustafa Usta 2 Julia D. Kenny 1 Peter J. Clements 1 Ingrid Pruimboom-Brees 1 Mike Aylott 0 Anthony M. Lynch 1 Cyrille A.M. Krul 3 0 Statistical Sciences Europe , GlaxoSmithKline, Park Road, Ware, Hertfordshire, SG12 0DP, UK 1 Safety Assessment, GlaxoSmithKline R&D, Park Road, Ware, Hertfordshire, SG12 0DP, UK 2 TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands 3 TNO, Utrechtseweg 48, 3704 HE Zeist, The Netherlands The Author 2012. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: . - An in vivo photomicronucleus test (MNT) using rat skin, the target organ for photoirritancy and carcinogenicity, was recently described. The assay was evaluated using fluoroquinolone (FQ) antibiotics with varying degrees of phototoxic potency (i.e. sparflocacin [SPFX], lomefloxacin [LOFX], ciprofloxacin [CIFX], levofloxacin [LEFX], gemifloxacin [GEFX] and gatifloxacin [GAFX]) using a solar simulator producing both UVA and UVB (ratio 23:1). Experiments were performed at The Netherlands Organisation for Applied Scientific Research (TNO) and GlaxoSmithKline (GSK) to investigate interlaboratory variability, including evaluation of phototoxicity (clinical signs), micronucleus induction and histopathology. The potency of micronuclei (MN) formation in rat skin induced by the FQs was SPFX=LOFX > CIFX=LEFX, however, MN induction was only statistically significant for SPFX and LOFX. In both laboratories, GEFX and GAFX did not increase the MN frequencies compared to the irradiated vehicle control. Signs of phototoxicity, including clinical and histopathological changes, were observed with SPFX and LOFX to a similar degree as the positive control, 8-methoxypsoralen. In addition, there were some clinical signs of phototoxicity seen with CIFX, LEFX, GEFX and GAFX, but not always in both laboratories for CIFX, GEFX and GAFX and when observed, these were considered only mild. Of these, only LEFX also showed histopathological changes. In all studies, photogenotoxic potency correlated with photocarcinogenic potential and moreover, photogenotoxicity was not observed in the absence of phototoxicity. The results of the TNO/GSK study indicate that the in vivo rat skin photoMNT may be a promising tool for detection of photoclastogencity and photoirritancy in the skin/eye in the same animal. Given the association between the MNT and cancer, the skin photoMNT may also provide a promising tool for the early detection of photocarcinogenesis and help bridge the gap in the existing photosafety testing paradigm. Introduction Exogenous compounds that reach the skin after topical application or following oral exposure, such as pharmaceuticals and personal care products, can be activated by solar radiation and may consequently contribute to adverse effects in the skin, such as photoirritation, photoinduced ageing or photocarcinogenicity (skin cancer) (13). As a result, photosafety testing has become a mandatory regulatory requirement for consumer products that both absorb light in the range of 290700 nm, and with (relevant) exposure in the skin or eyes. Details on the strategy and approaches for photosafety evaluation of products prior to review by regulatory authorities are described in guidance documents prepared by the European Medicines Agency (EMA) and the USA Federal Drug Agency (FDA) Center for Drug Evaluation and Research (CDER) (4,5). Testing may include evaluation of acute phototoxicity (photoirritation), photoallergy, photogenotoxicity and photocarcinogenicity (6). In terms of photogenotoxicity testing, the main objective is the early detection of the potential of a compound to induce tumours upon activation with ultra violet (UV) and/or visible light. Where considered necessary, a tiered approach to photosafety testing has been recommended, which includes an acute assessment of photo(geno)toxicity in vitro for hazard identification prior to in vivo assessments for risk characterization. The shortcomings of the current photosafety testing strategy are extensively described previously (7), including oversensitivity and the occurrence of pseudo-effects with in vitro assays, and the lack of availability of short-term in vivo photo(geno)-toxicity assays for additional evaluation of positive (or equivocal) results from in vitro assays. As a consequence, the number of false positive and need for unnecessary in vivo photocarcinogenicity studies is unacceptedly high. For these reasons, an in vivo photomicronucleus test (MNT) using rat skin, the target organ for photoirritancy and carcinogenicity, was developed, allowing relatively rapid evaluation of compounds with reduced numbers of animals compared to in vivo photocarcinogenicity studies. The choice of animal species and strain was based on the premise that many other toxicological and kinetic parameters are determined in rat and that by and large, the rat is used as the primary species for genotoxicity assessment in the pharmaceutical industry. As part of the assay development, several parameters were considered, including selection of UV irradiation modality and dose, optimization of skin harvest time after exposure for maximal micronucleus (MN) frequencies, characterization of 8-methxypsoralen (8-MOP) treatment for use as a positive control and the reproducibility of the method was demonstrated (see reference 7 for details). These studies indicated that the in vivo rat skin photoMNT may provide a promising assay for the assessment of both in vivo phototoxicity and photogenotoxicity, and an early prediction of photocarcinogenic liability in the target organ of interest. The present article describes an evaluation of the predictive capacity of the in vivo rat skin photoMNT using compounds of a single chemical class of phototoxins, the fluoroquinolone (FQ) antibiotics. The interaction of some FQ antibiotics with the mammalian topoisomerase II enzyme is responsible for their genotoxic potential in mammalian organisms (810). The antibacterial effect of FQs is due to their inhibition of the bacterial topoisomerase type II enzymes, such as bacterial gyrase. Type II topoisomerases are essential nuclear enzymes found in prokaryotic and eukaryotic cells that regulate the topological state of DNA during replication, transcription and repair. During the topoisomerase II cycle, the enzyme covalently binds to DNA and produces temporary double-strand breaks, thus creating a transient gate (cleavage complex) through which another DNA duplex can pass. After strand passage the break is ligated and the DNA structure is restored. Numerous compounds are known to disrupt the DNA breakagereunion cycle of mammalian topoisomerase II. This disruption during DNA transcription or replication can result in DNA strand breaks being exposed and this may lead to clastogenicity and/or cytotoxicity if the exposed DNA strand breaks are not repaired (11). In addition to (...truncated)