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Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.

In recent years genotoxicity testing has become more and more important in the process of early screening for potential development compounds. In the case that a pharmacologically interesting structure is found to be positive in an in vitro mutagenicity test a straightforward approach starts by sorting out what substructure is responsible for the activity observed in the test. The Ames test is a rapid, convenient test system which has been effectively used in structure-activity relationship studies for mutagenicity, since it can rapidly establish differences in the mutagenic action of isomers and chemical analogs. The lead compound with a benzodiazepine-like structure and close analogs exhibited weak, but unequivocal positive effects in the Ames test (strains TA1535 and TA 100) after metabolic activation by rat liver homogenate fraction (S9). To identify substances within this class of compounds devoid of mutagenic liability an extensive structure-activity investigation was undertaken. More than 50 compounds were tested in the two critical bacterial strains, using a standard plate incorporation and a preincubation modification. It quickly became evident that the benzodiazepine structure was not involved. First hints that the allyl side chain were responsible for the Ames activity had to be refined in a more complex, but clear-cut structure-activity relationship during the course of the experiments. It was shown that all compounds with an allyl side chain, independent of the heterocycle, but surprisingly also all compounds with a specific arrangement of the heteroatoms in the oxadiazole ring, showed positive effects in at least one strain. Based on these investigations it was possible to select pharmacologically active structures without mutagenic liability.

It has been commonly accepted that risk assessments of genotoxic chemicals are based on linear extrapolation methods. However, there is substantial evidence that some chemicals may be genotoxic only at high doses by mechanisms that do not occur at low doses, or only under specific conditions in genotoxicity assays, but are inactive at concentrations within the range of human exposure levels. There are a variety of possible mechanisms of thresholded genotoxicity, including disruption of cell division and chromosome segregation, inhibition of DNA synthesis, overloading of oxidative defence mechanisms, metabolism or plasma binding capacity, disturbances of metal homeostasis, cytotoxicity and physiological perturbations in in vivo assays. The degrees of evidence supporting the proposed mechanisms are variable and not all are sufficiently robust to be universally accepted as yet by the scientific community. However, a survey of industrial companies indicated that data have been accepted by some regulatory authorities indicating thresholds contributing to genotoxicity responses.

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., (Copyright 1999 Elsevier Science B.V.)

Induction of DNA damage as a consequence of exposure to UV light has been established as the major and still increasing cause of skin cancer. Absorption of the photon energy may be either directly by the DNA molecules (for wavelengths < 320 nm) or may be by endogenous or exogenous chemicals (sensitizers) with the potential of energy or electron transfer to DNA. Oxygen-mediated reactions (often called type II reactions) appear to be the most important mechanism since molecular oxygen is a good and abundant substrate for triplet excited sensitizers. Energy transfer to molecular oxygen is possible for wavelengths in the near UV and in the visible part of the solar spectrum since the energy of the excited oxygen molecule ((1)O2*) is comparatively low. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably psoralene and chlorpromazine derivatives have been established as photomutagens and the reaction mechanisms have been identified. The fluoroquinolone antibiotics have more recently been recognized as being photomutagenic. The type of DNA damage and the modulation by antioxidants indicate the involvement of reactive oxygen species (ROS) but other mechanisms are also reported at least for some derivatives. In routine genotoxicity studies we observed a photomutagenic activity of a compound under development as an anxiolytic agent in the Ames tester strain TA102 at 'normal laboratory illumination' conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The compound proved to be a potent (1)O2-producer. The finding led to termination of development but in the course of studies several structural analogues have been tested for which structure activity relationships will be described. The relevance of photogenotoxic properties of drugs for predicting adverse effects in man will be discussed.

L-Isohistidine and D,L-isohistidine, but not D-isohistidine, caused an increase of the number of mutant colonies in S. typhimurium strain TA100. Spontaneous and also sodium azide or 2-aminoanthracene induced mutant numbers were enhanced by L-isohistidine and by an isomeric mixture of D,L- and L-isohistidine. These effects could not be attributed to a growth-enhancing property. The colony probe hybridization procedure was used to investigate the effects of the histidines on the spontaneous and azide-induced spectra of the hisG46 allele in strain TA100. D,L-Isohistidine, but not the D-isomer, caused and increase of transitions (CCC-->CTC) and transversions (CCC-->CAC) in the spontaneous spectrum. Sodium azide alone induced a strong increase of CCC-->CTC transitions; combination with the D,L-isohistidine led to a further enhancement of this type of base substitutions, whereas with the L-isomer, no such effect was observed. This supports the hypothesis that the activity of D,L-isohistidine is probably not due to DNA-damaging properties, but rather to indirect mechanisms, such as enhancement of the infidelity of DNA replication and/or interference with DNA-repair or proofreading functions.

The ability of fluoroquinolones to cause light-induced adverse effects has been established in experimental studies and clinical observations. The formation of active oxygen species appears to be responsible for this activity. Photomutagenicity tests with bacterial, lower eukaryotic and mammalian cells were performed with three fluoroquinolones (Fleroxacin, Ciprofloxacin and Lomefloxacin). After concomitant irradiation with simulated solar light (with a reduced UVB component), weak increases in the number of revertants were observed in Salmonella typhimurium TA104 and TA100. No photomutagenic activity was detected in Saccharomyces cerevisiae D7. In the chromosomal aberration (CA) test with Chinese hamster V79 cells the number of aberrant metaphases was markedly increased. In the Comet assay with mouse lymphoma cells, evidence of extensive DNA breakage was obtained. All three compounds showed similar potencies in the Comet and Ames assays while there was a clear gradation of potencies in the CA assay (Lomefloxacin > Fleroxacin > Ciprofloxacin), which conformed with reports on the relative potencies regarding phototoxicity. The oxygen radical scavengers catalase, superoxide dismutase and N, N'-dimethylurea modulated the photoclastogenicity and phototoxicity but had no influence on the effects in the Comet and Ames tests. It thus appears that different kinds of mechanism are responsible for toxicity and clastogenicity on the one side and DNA breakage and gene mutation on the other side. Pre-irradiation of the test articles did not lead to enhanced genotoxicity, indicating the involvement of very short lived genotoxic agents. The results endorse the advice to avoid excessive light exposure during antibiotic therapy with fluoroquinolones.

A number of structurally very diverse compounds which cause weak positive effects in the Ames test by evident or suspect irrelevant mechanisms is discussed. As a unifying observation we describe synergistic effects in combination with known mutagens in the responsive strains and comutagenic effects in initially unresponsive strains. We argue that the compounds enhance the formation of spontaneous (or mutagen-induced) revertant colonies by test-specific mechanisms likely to be of no relevance to multicellular eukaryotic organisms rather than possessing intrinsic genotoxic (i.e. DNa-damaging) properties in the Ames test.

The genotoxic potency of certain classes of topoisomerase II poisons is correlated with their affinity to the topoisomerase protein rather than with the presence of 'classical' structural alerts for DNA reactivity: bacterial topoisomerase II poisons (specifically named gyrase inhibitors) are highly genotoxic in prokaryotic systems; mammalian topoisomerase II poisons are potent mutagens/clastogens in eukaryotic systems. Studies with bacterial, lower eukaryotic and mammalian genotoxicity tests were performed to draw structure-activity conclusions and address risk-benefit considerations for the class of quinolone gyrase inhibitors. All 17 gyrase inhibitors investigated in this study showed genotoxic activity in Salmonella typhimurium strain TA102 and the SOS test. The genotoxic and the toxic activities increased in a highly parallel fashion from the parent compounds, nalidixic acid and oxolinic acid, to the new generation fluoroquinolones. Generally, the most potent fluoroquinolones also show clear-cut positive effects in eukaryotic test systems, although at concentrations 100-1000-fold higher than those effective in bacteria and also 100-1000-fold higher than the minimal genotoxic concentrations of antitumour topoisomerase II inhibitors (ellipticine, teniposide, mAMSA) used as reference compounds. However, subtle structural modifications of the quinolones can strongly diminish the preferential genotoxicity in the prokaryotic test systems.

Two in vitro genotoxicity tests have been adapted to the evaluation of photomutagenic activity of test compounds. The study was initiated to obtain an experimental basis relating to newly proposed guidelines of the EC which request the screening of UV-absorbing compounds, for example, those employed in sunscreen preparations, for their photomutagenic potential. The well established photomutagens 8-methoxypsoralen and chlorpromazine were used to define relevant test protocols. The compounds were evaluated with the Ames test and the Saccharomyces cerevisiae D7 test for gene conversion. The influence of various parameters such as UV light sources, spectral composition, UV sensitivity of the test systems, absorbance by test materials and different exposure conditions is indicated. Two exemplary screening experiments with cosmetic ingredients are presented. Both test systems can be employed for the evaluation of compounds for photomutagenic activity although the standard excision-deficient strains of S. typhimurium pose problems because of their high UV sensitivity. The present experience in this complex field suggests that rigid test protocols and a restrictive test battery would be inadequate.

The liver carcinogen phenobarbital (PB) causes a weak but reproducible increase of the mutant frequency in the Ames test, strain TA1535, without S9. Since there is no obvious chemical basis for a "DNA reactivity" of this compound experiments were performed to obtain information about possible indirect mechanisms of enhancing the number of spontaneous mutant colonies. In the course of the study strong synergistic and comutagenic effects of PB when given in combination with Na-azide or 2-aminoanthracene (2AA) were observed. Not only TA1535 but the complete set of tester strains was responsive. However, PB did not enhance the effects of other mutagens such as 4-nitroquinoline N-oxide or 2-nitrofluorene. It is argued that in strain TA1535 the fixation and expression of spontaneously occurring DNA lesions is amenable to modulation by PB similar to that of Na-azide or 2AA induced lesions. Thus in the usual sense, PB is not genotoxic in the Ames test. Methapyrilene, another liver carcinogen with an assumed nongenotoxic mode of action, showed almost identical properties in these experiments.

Our objective is to summarize the published data obtained with a recently developed tester strain suitable for the detection of chromosomal malsegregation in yeast. Results from 25 papers were reviewed in which numerical data for 111 chemicals tested in Saccharomyces cerevisiae D61.M are reported (a total of 316 independent tests; 279 acceptable, 37 not meeting our criteria). Of the 111 compounds analyzed 43 compounds are positive for chromosomal malsegregation, 56 compounds are negative and 12 compounds do not meet our criteria for acceptance (inconclusive). Of the 43 compounds judged positive 5 (acetone, acetonitrile, benzonitrile, ethylacetate and propionitrile) were only positive using a cold interruption protocol. Recommendations are made for standardization of methods and protocols for screening purposes. Finally, a comparison with in vitro tubulin assembly data using mammalian tubulin is presented.

The state of aneuploidy test methodology was appraised by the U.S. Environmental Protection Agency in 1986 in analyzing published data. In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells. We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation. Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation. Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells. No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S. cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml). A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9. Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA. We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events). Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).

We tested the 10 known or suspected spindle poisons (colchicine, econazole nitrate, chloral hydrate, hydroquinone, diazepam, thiabendazole, cadmium chloride, thimerosal, pyrimethamine and vinblastine) of the coordinated EEC programme for induction of aneuploidy with the in vitro porcine brain tubulin assembly assay. The influence of the compounds on different parameters [lag-phase, polymerization velocity, endabsorption (steady-state level), reversibility, influence on disassembly at 4 degrees C] was evaluated. Colchicine [IC30 (30% inhibition concentration): 0.002 mM), vinblastine (IC30: 0.002 mM), thimerosal (IC30: 0.03 mM), thiabendazole (IC30: 0.5 mM) and chloral hydrate (IC30: 60 mM) led to an inhibition of tubulin assembly in vitro. No influence on the steady-state level was obtained with econazole nitrate (up to 0.1 mM), diazepam (up to 2.5 mM), cadmium chloride (up to 1 mM), pyrimethamine (up to 1 mM) and hydroquinone (up to 25 mM), the highest dose tested being limited either by precipitation or by reaching the maximal solubility of the compound in the solvent used. Diazepam enhanced the lag-phase and slightly reduced the polymerization velocity dose-dependently; however, all the treated test mixtures reached the same end absorption levels as the control. The influence on the disassembly process was studied at 4 degrees C. Microtubules treated with colchicine, econazole nitrate, diazepam, thiabendazole, cadmium chloride, thimerosal and pyrimethamine reached the same end absorption level after disassembly as the untreated control. Chloral hydrate reduced the disassembly rate but the end absorption of the control was not reached, the 30% reduction concentration being 0.25 mM. Hydroquinone at very high doses (greater than 10 mM) stimulated the disassembly process.(ABSTRACT TRUNCATED AT 250 WORDS)

We tested nine (cadmium chloride, chloral hydrate, colchicine, diazepam, econazole nitrate, hydroquinone, pyrimethamine, thiabendazole, thimerosal) of the 10 known or suspected spindle poisons of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation system). Mitotic malsegregation of chromosome VII was induced by chloral hydrate, thiabendazole and thimerosal. Diazepam, colchicine, cadmium chloride, econazole nitrate, hydroquinone and pyrimethamine revealed no induction of chromosomal malsegregation.

Bavistan is known to be a potent inducer of chromosome malsegregation in Saccharomyces cerevisiae. The influence of different factors on the induction of chromosome malsegregation in S. cerevisiae D61.M was investigated. With both standard protocols used (16 h overnight incubation and cold treatment protocol) bavistan, in a concentration range of 2.5-20 micrograms/ml, induced malsegregants to the same extent. The frequencies of malsegregants obtained were not influenced by the plating volume used on selective medium. Induction of malsegregants and toxicity became stronger with increasing supplementation of the incubation medium with yeast extract and peptone. The effects of bavistan on chromosome malsegregation were more pronounced at 28 degrees C--the normal temperature for yeast growth--as compared to 33 and 37 degrees C. A study of the time dependence of the induction of chromosome loss showed that malsegregants can already be detected after 8 h and 1.5 h (second incubation period) using the incubation protocols without and with cold treatment, respectively. To clarify whether a selection towards malsegregants occurs, the growth of mixed cultures of red, cycloheximide-sensitive cells and white, cycloheximide-resistant, leucine-auxotrophic cells prepared at different ratios was compared. A strong selection towards red cells and against the malsegregants was observed. In addition, bavistan was tested for genotoxic activity in Salmonella (Ames test) and in yeast S. cerevisiae D7. No mutagenic activity was detected using S. cerevisiae D7 (gene conversion, reverse mutation, mitotic crossing-over) with and without rat-liver S9. In contrast bavistan induced histidine revertants in the frameshift strains TA1537, TA1538, TA97 and TA98 of Salmonella typhimurium after addition of an exogenous metabolic activation system.

Mutagenic products can be formed during the processing of food and especially as a result of heat treatment. Direct acting mutagenic activity was found in extracts of instant coffee and roasted coffee beans using Salmonella typhimurium TA100 in vitro. The mutagenic activities of the four pure coffee varieties examined (Coffea arabica Santos, Coffea arabica Columbia, Coffee robusta Indonesia, Coffee robusta Camerun) were within the same range. Twenty milligrams per plate freeze-dried powder prepared from aqueous roast coffee extracts induced between six and ten times the number of revertants found in the negative controls. Green coffee beans had no mutagenic activity. Mutagenicity increased with roasting time up to 4 min in the Probat drum roaster and then remained constant (i.e. no further increase after 8 min, the time normally used to roast coffee). The genotoxic compounds were quickly formed at temperatures below 220 degrees C (in normally roasted coffee the beans must reach a temperature of 220 degrees C). Mutagenic activity was independent of the roasting procedure (Jetzon procedure v. Probat drum roaster).