Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop.

The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.

O-phenylphenol and its sodium and potassium salts: a toxicological assessment.

Ortho-phenylphenol (OPP) and its sodium (SOPP) and potassium (POPP) salts are used as fungicides and disinfectants. Due to the widespread use of especially OPP and SOPP, the potential for consumer exposure and some "critical" findings the toxicological database is quite extensive and complex. In experimental animals toxicity after single oral and dermal administration of these compounds is low. For the skin and mucous membranes, OPP has to be considered as irritating, and SOPP and POPP as corrosive. A large number of chronic toxicity and reproduction studies did not show any indication of oestrogen-like or other endocrine effects of OPP in the mammalian organism. No teratogenic effects were observed after the administration of OPP or SOPP in rats, mice, and rabbits. In two-generation studies in rats, OPP did not affect reproduction. The available data do not suggest a relevant potential for immunotoxic properties. The administration of high dietary concentrations of OPP to mice up to 2 years induced hepatocellular changes indicative of adaptations to metabolic demands, zonal degeneration, focal hepatocellular necrosis, and/or pigmentation of the liver. Only in male mice of one study, using a strain prone to develop hepatocellular tumors at high spontaneous incidences, the incidence of hepatocellular adenomas was increased. The incidence of hepatocellular carcinomas was not affected by treatment. The urothel of the urinary bladder (at very high doses also of the renal pelvis and the papilla) is the main target tissue after the repeated oral exposure of rats. The changes initially consist of increased mitosis, followed by simple epithelial hyperplasia, developing to a papillary and/or nodular form, later on to papillomas and transitional carcinomas. Crystals or stones in the bladder do not play a decisive role in this cascade. SOPP is more effective than OPP in this respect. Male rats are much more sensitive than females. In mice, hamsters, guinea pigs, and dogs, urothelial lesions do not develop even at very high oral dose levels. The findings in rats explain why there is a large genotoxicity/mutagenicity data base not only for OPP and SOPP but also for their metabolites on nearly all kinds of endpoints/targets. The weight of evidence suggests that genotoxicity of OPP/SOPP or their metabolites does not play a decisive role for the carcinogenicity at the urothel. Among them are lack of DNA binding of OPP to the rat bladder epithelium, the differences between OPP and SOPP, between male and female rats, between rats and mice (despite roughly comparable toxicokinetics), as well as the fact that tumors develop only at dose levels inducing hyperplasias. In addition, the strong dependence of the incidence and severity of the nonneoplastic and neoplastic bladder changes on urinary pH values (modified by feeding of ammonium chloride or sodium hydrogen carbonate) is consistent with the hypothesis of a nongenotoxic mode of action. Finally, there is no correlation between the urinary concentration of OPP or its metabolites and the incidence of hyperplasias/tumors in the urinary bladder. Both tumorigenic effects in rats and male mice are considered to represent high-dose, sex- and/or species-specific phenomena, based on nongenotoxic mechanisms of action and therefore allow the conclusion that the conventional margin of safety approaches are appropriate when assessing the risk of applications of OPP and its salts.

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.

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.

[In vitro methods for phototoxicity and photocarcinogenicity testing of drugs]. / In vitro Methoden zur Prüfung von Arzneimitteln auf photogenotoxische/photokanzerogene Eigenschaften.

Phototoxicity is an acknowledged property of some UV and/or visible light absorbing substances some of which are used as pharmaceuticals or in cosmetic preparations. In recent years attention has been called upon the fact that toxic intermediates that are generated upon photoactivation of a substance can also lead to DNA damage. Such damage may lead to mutated/initiated skin cells which in turn can contribute to an elevated skin cancer risk. The method of choice to test for photo-related skin carcinogenesis is a 1-year study in genetically hairless mice in which the formation of skin papilloma and their latency time are assessed. Here, in vitro test approaches to test for photogenotoxicity can be used in a tiered assessment approach asking the use of in vitro genotoxicity tests for prediction of rodent/human carcinogenicity. In the past few years some effort has been put into the evaluation for such systems, in particular standard test protocols have been generated for the in vitro photo-micronucleus test and the in vitro photo-comet assay with Chinese hamster V79 cells. The data that have been produced so far show promising results regarding the implementation of these systems in a tiered approach for photocarcinogenicity assessment of UV- and/or visible light absorbing substances but the systems will have to be validated in further collaborative studies.

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin.

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.

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.

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.

Genotoxicity testing of biotechnology-derived products. Report of a GUM task force. Gesellschaft für Umweltmutationsforschung.

Various aspects of genotoxicity testing of biotechnology-derived products are discussed based on information gathered from a questionnaire which was sent to about 30 predominantly European companies. Feedback was received from 13 companies on 78 compounds, mostly recombinant proteins but also on a number of nonrecombinant proteins, which had been assessed for genotoxicity in a total of 177 tests. Four of the 78 compounds appeared to elicit reproducible genotoxic effects. For one of these compounds, the activity could be related to a nonpeptidic linker molecule. No scientifically convincing rationale for the other three compounds could be established, although, at least for two compounds, their activity may be connected with the enzymatic/hormonal activity. In addition to the survey, published reports on genotoxicity testing of biotechnology products were reviewed. The data are discussed relative to whether genotoxicity testing is a valuable exercise when assessing potentially toxic liabilities of biotechnology-derived compounds. It is concluded that genotoxicity testing is generally inappropriate and unnecessary, a position which is in accordance with the available guidelines addressing this area. For the 'average' protein, electrophilic reactions are difficult to envision. Indirect reactions via DNA metabolism and growth regulation seem possible for only very specific proteins such as nucleases, growth factors, cytokines. No information on testing of different types of biotechnology-derived products (e.g., ribozymes, antisense-oligonucleotides, DNA vaccines) has been received in the questionnaires. Discussion of their potential to cause genotoxic changes was based on literature reports. Even for those products for which concerns of genotoxic/tumourigenic potential cannot be completely ruled out, e.g., because of their interaction with DNA metabolism or proliferation control, the performance of standard genotoxicity assays generally appears to be of little value. All information, including also information on the occurrence of genotoxic impurities, has been utilized to formulate a decision tree approach for the genotoxicity testing of biotechnology-derived products.

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.

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.

Definition of a new intralaboratory limit value for a positive UDS assay in vitro.

In our laboratory, we routinely perform the unscheduled DNA synthesis (UDS) assay in vitro using rat primary liver cells and autoradiographical evaluation. The fixed limit of +5 nuclear net grains (NNG) for a positive UDS response no longer seems to be appropriate because of the advanced techniques that are now used. Considering our historical data (mean vehicle control NNG of 45 experiments = -1.5; highest observed mean NNG value in 126 control slides = +1.63 NNG) we will use a mean value of +2 NNG as our new intralaboratory limit value for a positive UDS assay, independent of the statistical evaluation. Experiments with a mean NNG count in the vehicle control of > +0.5 will be rejected as invalid and repeated since mean values of > +0.23 NNG were never reached. Additionally, the NNG data obtained will be subjected to a newly proposed statistical method which includes both the actual and the mean historical control NNG value. First, the sum of the actual and the mean historical control value is divided by two. Secondly, the calculated control value is pairwise compared with the treated groups by the one-sided Dunnett's test. This might be a helpful tool to evaluate a weak or equivocal UDS response.

Cells of different tissues for in vitro and in vivo studies in toxicology: Compilation of isolation methods.

An advantage of using freshly isolated intact cells of different organs in toxicology is that they reflect more closely the in vivo situation than do long-term cultures. In vitro, primary cells provide the possibility of determining cell-specific xenobiotic metabolism, in the absence of artificial extracellular activation systems, which may result in cytotoxic and genotoxic effects. After in vivo exposure of animals to xenobiotics, isolated primary cells can be studied to elucidate toxicokinetic effects. In the review presented here, selected methods are described for isolating cells with high viability from pig liver and avian embryonic liver, and from the nasal cavity, lungs, kidneys, gastro-intestinal tract, urinary bladder, testes and thymus of the rat. Two techniques for preparing rat lymphocytes are also described. Cell isolation may be initiated with an in situ perfusion to clear the organ of blood. Steps to loosen cell-to-cell contacts and to digest the intercellular connective material may then follow. Also, in situ digestion may be performed, as described for the epithelial cells from different mucosal tissues. Following initial digestion, a single-cell suspension is prepared by tissue mincing and a second digestive step with proteolytic enzymes. Frequently used digestive enzymes are collagenase (types I, IV and P; from Clostridium histolyticum), trypsin and proteinase K. Follow-up filtration is usually required to remove undigested material. The quantities and viabilities of the harvested cells vary with the organ of choice and the procedure used; the values obtained are stated.