Susanne Brendler-Schwaab

Photochemical genotoxicity: principles and test methods. Report of a GUM task force.

In recent years, assessing the photogenotoxic potential of a compound became an issue for certain drugs and cosmetical products. Therefore, existing methods performed according to international guidelines (e.g. OECD guidelines) were adapted to the use of concurrent UV-visible (UV-Vis) light irradiation for the assessment of photomutagenicity/photogenotoxicity. In this review, photobiological bases of the processes occurring in the cell after irradiation with UV- and/or visible (vis)-light as well as a compilation of testing methods is presented. Methods comprise cell free investigations on naked DNA and in vitro methods, such as the photo-Ames test, the photo-HPRT/photo-mouse lymphoma assay (MLA), the photo-micronucleus test (MNT), the photo-chromosomal aberration test (CA) and the photo-Comet assay. A compilation of the currently available international literature of compounds tested on photogenotoxicity is given for each method. The state of the art of photogenotoxicity testing as well as the rational for testing are outlined in relation to the recommendations reached in expert working groups at different international meetings and to regulatory guidance papers. Finally, photogenotoxicity testing as predictor of photocarcinogenicity and in the light of risk assessment is discussed.

The application of the micronucleus test in Chinese hamster V79 cells to detect drug-induced photogenotoxicity

Recent reports on the photochemical carcinogenicity and photochemical genotoxicity of fluoroquinolone antibacterials led to an increasing awareness for the need of a standard approach to test for photochemical genotoxicity. In this study the micronucleus test using V79 cells was adapted to photogenotoxicity testing. Results of using different UVA/UVB relationships enabled us to identify a suitable irradiation regimen for the activation of different kinds of photosensitizers. Using this regimen, 8-methoxypsoralen and the fluoroquinolones lomefloxacin, grepafloxacin and Bay Y 3118 were identified to cause micronuclei and toxicity upon photochemical activation. Among the phenothiazines tested, chlorpromazine and 2-chlorophenothiazine, were positive for both endpoints, whereas triflupromazine was only slightly photoclastogenic in the presence of strong phototoxicity. Among the other potential human photosensitizers tested (oxytetracycline, doxycycline, metronidazole, emodin, hypericin, griseofulvin), only hypericin was slightly photogenotoxic. Photochemical toxicity in the absence of photochemical genotoxicity was noted for doxycycline and emodin. With the assay system described, it is possible to determine photochemical toxicity and photochemical genotoxicity concomitantly with sufficient reliability.

Considerations on photochemical genotoxicity: Report of the International Workshop on Genotoxicity Test Procedures Working Group

Recent toxicological observations have caused concern regarding the need to test, for example, pharmaceuticals and cosmetic products for photochemical genotoxicity. The objective of this report is to give assistance on how to adapt existing test methods to investigate the potential of light‐absorbing compounds to induce genotoxic effects on photoactivation. In general, the Organization for Economic Co‐Operation & Economic Development (OECD) draft guideline on in vitro phototoxicity testing served as a basis for consideration. Concomitant exposure of the cells to the test compound and solar simulated light was considered appropriate as the initial, basic test condition. Optimization of the exposure scheme, e.g., a change of the irradiation spectrum, might be indicated depending on the initial test results. Selection of test compound concentrations should be based on results obtained with the dark version of the respective test system but might have to be modified if phototoxic effects are observed. Selection of the irradiation dose has to be performed individually for each test system based on dose‐effect studies. The irradiation should induce per se a small, reproducible toxic or genotoxic effect. The report includes a specification of necessary controls, discusses factors that might have an impact on the irradiation characteristics, and gives a rationale for the omission of an external metabolic activation system. It also addresses the question that physicochemical and pharmacokinetic properties might trigger the need to test a chemical for photochemical genotoxicity. Relevant experimental observations are presented to back up the recommendations. The working group did not reach a consensus as to whether a single, adequately perfomed in vitro test for clastogenicity would be sufficient to exclude a photogenotoxic liability or whether a test battery including a gene mutation assay would be needed for product safety testing regarding photochemical genotoxicity. Environ. Mol. Mutagen. 35:173–184, 2000

Ciprofloxacin: in vivo genotoxicity studies.

The fluoroquinolone ciprofloxacin is widely used in antimicrobial therapy. It inhibits the bacterial gyrase and in high concentrations in vitro also the functionally related eukaryotic topoisomerase-II, which resulted in genotoxic effects in several in vitro tests. In order to evaluate the relevance of these findings, ciprofloxacin was tested in vivo for genotoxic activity using the following test systems: micronucleus test in bone marrow of mice, cytogenetic chromosome analysis in Chinese hamster, dominant lethal assay in male mice and UDS tests in primary rat and mouse hepatocytes in vivo. These results are compared with already published in vitro and in vivo studies with ciprofloxacin. All in vivo genotoxicity revealed no genotoxic effect for ciprofloxacin. In addition, ciprofloxacin was found to be non-carcinogenic in two rodent long-term bioassays. Therefore, ciprofloxacin is considered to be safe for therapeutic use.

Use of the photo-micronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity.

Photochemical genotoxicity can be detected using appropriately adapted versions of most of the standard in vitro genotoxicity assays. The most sensitive approach to detect potentially photogenotoxic agents seems to be the investigation of DNA damage (DNA strand breakage, chromosomal aberrations, micronuclei) in mammalian cells in vitro. In a previous paper, we proposed the use of the micronucleus assay in Chinese hamster V79 cells for this purpose. This assay was found suitable to detect various photogenotoxic compounds with different photoactivation mechanisms. In order to extend the experimental experiences with this assay, we present here further data from a screening mode testing of 16 different potential photosensitizers. The photoclastogenic and photocytotoxic potential of the compounds was investigated concomitantly. So far, all substances detected in the photo-micronucleus assay as photogenotoxins also exhibited photocytotoxic properties but not vice versa. Among the compounds tested in the present study, tiaprofenic acid, 5-MOP, angelicin, nitrazepam, bendroflumethiazide, and dacarbazine were photogenotoxic and photocytotoxic. Further, 6-mercaptopurine, a metabolite of azathioprine was positive for both endpoints, whereas azathioprine was found negative. Azathioprine seems to be an example of a compound which lacks photo(geno)toxic properties in vitro but may be converted to a photosensitizer by enzymatical metabolization. With the results obtained in this study, the data base for the photo-micronucleus assay was extended to 35 compounds, which were tested using the same protocol and the same irradiation conditions. The photogenotoxicity results of all these compounds are summarized and discussed in correlation to their different photoactivation mechanisms, photocytotoxicity and photocarcinogenicity.

Prevalidation of a Rat Liver Foci Bioassay (RLFB) Based on Results from 1600 Rats: A Study Report

A rat liver foci bioassay (RLFB) based on an initiation-promotion protocol employing preneoplastic foci of altered hepatocytes (FAH) as an endpoint, was prevalidated in 5 different laboratories. FAH were identified by immunohistochemical demonstration of glutathione-S-transferase (placental form, GSTP) and by staining with hematoxilin/eosin (H&E), and their area fraction was quantified morphometrically. The four model hepatocarcinogens N-nitrosomorpholine, 2-acetylaminofluoren, phenobarbital, and clofibrate were selected according to characteristic differences in their presumed mode of action, and tested in a total of 1,600 male and female rats at 2 different dose levels. The chemicals were found to differ characteristically in their potency and dose-response relationship to induce FAH when given alone or when administered following initiation with diethylnitrosamine. The interlaboratory variation was small for results obtained with the GSTP-stain and somewhat larger with respect to H&E. The assessment of the carcinogenic potential of the four chemicals by the different laboratories was in the same range and the nature of their dose-response relationships did not differ essentially between laboratories. Our results suggest that this RLFB is a sensitive bioassay, providing potentially valuable information for risk assessment including the classification of carcinogenic chemicals according to their mode of action.

A new method for the enrichment of single renal proximal tubular cells and their first use in the comet assay

A protocol was developed to isolate and enrich single renal proximal tubular cells, performing the following steps: in situ kidney perfusion; isolation of renal tissue pieces by collagenase digestion; selective enrichment of proximal tubular fragments by Percoll gradient centrifugation; and isolation of single proximal tubular cells by digestion of proximal tubular fragments with trypsin. The mean enrichment rate, determined by the glucose-6-phosphatase staining method, was 78.9% with a mean cell viability of 93.8%. After modification of the comet assay protocol, genotoxicity in proximal tubular cells could be investigated. A dose-dependent genotoxic effect of ethyl methanesulphonate in these cells was proven.

Differences in tissue distribution of iron from various clinically used intravenous iron complexes in fetal avian heart and liver.

Nanomedicines are more complex than most pharmacologically active substances or medicines and have been considered as non-biological complex drugs. For nanomedicines pivotal pharmacokinetic properties cannot be assessed by plasma concentration data from standard bioequivalence studies. Using intravenous iron complexes (IICs) as model we show that fetal avian tissues can be used to study time dependent tissue concentrations in heart and liver. Clear differences were found between equimolar doses of sucrose, gluconate or carboxymaltose coated iron particles. The range in tissue iron concentrations observed with these clinically widely used IICs provides an orientation as to what should be acceptable for any new IICs. Moreover, sensitivity of the experimental model was high enough to detect a 20% difference in tissue iron concentration. For the authorization of generic products under Article 10 (1) of Directive 2001/83/EC a plasma concentration of an active substance in the range of 80%-125% versus the reference product is usually considered acceptable. Based on its high discriminatory sensitivity this method was used to support a positive marketing authorization decision for a generic nanomedicine product.

Carcinogen-induced Mitochondrial DNA Damage in the In Ovo Model

After experimental exposure of turkey eggs in an in ovo model, the induction of preneoplastic liver lesions (Enzmann et al., 1992and 1995a) and alterations of the mitochondrial (mt) DNA of embryonic turkey livers (Enzmann et al., 1995b) could be demonstrated, showing the sensitivity and usefulness of this short-termed and inexpensive system for carcinogenicity testing. Chemically induced modification of mtDNA may be an important indicator of the carcinogenic potential of substances, as the mt genome may display a higher sensitivity to DNA damaging effects compared to nuclear DNA. To characterize mtDNA damages in ovo, application of diethylnitrosamine (DEN) was performed onto the chorioallantoic membrane (CAM) of the avian embryo. First, the distribution of a model substance after CAM application was measured by autoradiography. MtDNA damage after DEN exposure was demonstrated by gel electrophoresis of isolated mtDNA. Nitrosamine treatment induced a dose-dependent change of mtDNA conformation from supercoiled to relaxed shape, pointing to a possible induction of single-strand breaks.

Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo.

In the first international guideline addressing the unscheduled DNA synthesis (UDS) assay in vivo (OECD guideline no. 486, adopted July 1997) only the genotoxic liver carcinogen N-nitrosodimethylamine (NDMA) is proposed as positive control for the short sampling time. Since NDMA is extremely volatile, alternative positive controls should be identified to facilitate handling and reduce exposure risk during routine testing. At Bayer AG and at RCC-CCR GmbH, the genotoxic but non-volatile dimethylhydrazine (DMH; as dihydrochloride) was used instead as positive control in livers of Wistar rats and to a limited extent of NRMI mice after 2-4h exposure. As shown by the data presented in this paper DMH induced a positive result in a total of 21 UDS in vivo studies over a period of 7 years. A negative result was never seen for DMH. Due to these results DMH was proven to be a suitable and reliable positive control in the UDS assay in vivo. Consequently, DMH should be considered as positive control for the short sampling time in the next issue of OECD guideline no. 486.