Bacterial mutagenicity screening in the pharmaceutical industry.

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development. This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Comprehensive assessment of the photomutagenicity, photogenotoxicity and photo(cyto)toxicity of azulene.

The polycyclic aromatic hydrocarbon azulene and its naturally occurring derivative guaiazulene (1,4-dimethyl-7-isopropylazulene) are known to absorb light in the UV-vis region of the spectrum. Both compounds were reported to be mutagenic in the Salmonella typhimurium bacterial mutagenicity assay (Ames test) in strain TA102, and to cause DNA damage in the comet assay in vitro upon exposure to UVA light. In contrast, another study reported a photoprotective effect in vitro of guaiazulene. We present here a comprehensive assessment of the photo(cyto)toxicity (3T3 fibroblast Neutral Red uptake test), the photomutagenicity (Ames test) and photogenotoxicity (comet assay and micronucleus test in L5178Y cells in vitro) of azulene. In the Ames test, the mutagenicity of azulene was assessed in the presence and absence of UV light by use of the Salmonella strains TA102, TA104, TA2638 and E. coli WP2. Azulene was irradiated before being plated with bacteria (pre-irradiation), or concomitantly with the bacteria either after plating or while in suspension. Guaiazulene was included in some of the experiments. Neither in the photo-Ames test nor in the other photogenotoxicity tests, azulene or guaiazulene showed any photomutagenic or photogenotoxic activity. Weak photo(cyto)toxicity (estimate of PIF≥1.67) was observed with azulene in the 3T3 NRU test, the Alamar Blue test and the relative cell count, which may be due to the generation of reactive oxygen species, as reported recently.

Photochemical mutagenesis: examples and toxicological relevance.

Induction of DNA damage as a consequence of exposure to UV light has been established as the major cause of skin cancer. DNA molecules absorb photon energy directly for wavelengths <320 nm, and lead to well-characterized mutagenic DNA damage. Alternatively, endogenous or exogenous chemicals (sensitizers) may absorb light with the potential of subsequent energy or electron transfer, and lead indirectly to DNA damage. A few light-absorbing pharmaceuticals have long been known to cause photo(geno)toxic effects. Notably, psoralen 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 for, at least, some derivatives. In routine genotoxicity studies, we observed the photomutagenic activity of a compound (Ro 19-8022) under development as an anxiolytic agent in the Ames tester strain TA102 under normal laboratory illumination conditions. Further investigations showed strong photogenotoxic activity in tests for gene mutations and chromosomal aberrations in mammalian cells. The finding led to the termination of drug development. Another example of a pharmaceutical for which photogenotoxic properties were observed during development is Ro 47-7737, a bisquinoline derivative of the antimalaria compound chloroquine. Also in this case, the photochemical reactivity contributed to the termination of the development process. The risk/benefit assessment for the described compounds has to take into account the human exposure situation, for example, the ability to avoid light exposure during treatment. Consideration of photochemical mutagenesis is specifically important for sunscreen ingredients. The active components of sunscreen lotions are efficient UV absorbers. Consequently, they reduce the genotoxicity of UV light and thus may be considered antimutagens. However, photodegradation to reactive molecules or energy transfer to DNA is possible, in principle, as has been reported for para-aminobenzoic acid (PABA).

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.

The photomutagenicity of fluoroquinolones and other drugs.

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.

Review of the genotoxic properties of chlorpromazine and related phenothiazines.

Chlorpromazine and related phenothiazine drugs have been used in human and veterinary medications for more than 40 years, predominantly as psychotropic agents. Genotoxicity reports are in many cases of relatively antiquated test design. Overall there appears to be no genotoxic activity associated with these drugs when tested under standard conditions. Limited evidence for the potential to form mutagenic nitrosation products and some indication for the ability to modulate the genotoxic action of various mutagens have been presented in the literature. UV irradiation of chlorpromazine and other chlorinated derivatives produces reactive free radicals which possess DNA damaging properties. Induction of gene mutation and chromosomal aberrations have been observed in appropriately designed photomutagenesis experiments. Enhancement but also reduction of UV induced skin tumour formation by chlorpromazine have been found. The decisive factor for the discrepant actions has not been recognized. It is clearly advisable to avoid extensive UV exposure during therapy with these drugs.

The photomutagenicity of fluoroquinolones in tests for gene mutation, chromosomal aberration, gene conversion and DNA breakage (Comet assay).

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.

Synergistic/comutagenic action in the Ames test as an indication of irrelevant positive findings.

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.

Genotoxicity of 17 gyrase- and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems.

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.

Evaluation of the micronucleus test in vitro using Chinese hamster cells: results of four chemicals weakly positive in the in vivo micronucleus test.

A rapid and simple procedure for the micronucleus test (MNT) in vitro using Chinese hamster ovary (CHO) cells was established in our laboratory. The assay is intended to quickly screen chromosomal aberrations in vitro within the framework of industrial genotoxicity studies. To test the sensitivity of the assay in the experiments described here, four substances, classified as noncarcinogens but reported as weak inducers of micronuclei (MN) in bone-marrow cells of mice, were evaluated in the MNT in vitro. Of the four compounds, ascorbic acid, phenol, and 2,6-diaminotoluene proved to be genotoxic in the MNT in vitro. Titanium dioxide, which could not be dissolved in the culture medium, did not induce MN. The MNT in vitro proved to be quick and relatively simple and to yield highly reproducible results when testing the four chemicals.

Normal chromosomal aberration frequencies in peripheral lymphocytes of healthy human volunteers exposed to a maximum daily dose of paracetamol in a double blind trial.

Paracetamol (acetaminophen) has been examined for mutagenic potential in numerous studies: gene mutation tests consistently gave negative results while in vitro chromosomal aberration tests showed equally consistently positive effects. In vivo studies for chromosome breaking activity gave clearly negative, equivocal or weakly positive results. In particular two reports have indicated that human volunteers taking a maximum daily dose of paracetamol (3 x 1000 mg over 8 h) exhibited significantly elevated frequencies of chromatid breaks in their peripheral lymphocytes 24 h later. In the one study evaluating the time course, levels returned to normal between 3 and 7 days later. We performed a carefully controlled double-blind study in which volunteers were pre-screened for normal liver function, they all were non-smoking and their diet and environmental exposures were controlled during the study. Cell-cycle kinetics were monitored and paralleled and a placebo group was included. Although a larger number of cells than in the other studies was analysed we were unable to reproduce their findings. No significant increases in structural chromosome aberrations (CA) were found either when the paracetamol group (male, female or both) post-dosing values were compared with pre-dosing values, or when treated groups at any sampling time were compared with the placebo groups. There was not even any evidence that individuals responded to the clastogenic potential of paracetamol or that a group response may have been masked by non-responders. In conjunction with the recently published results of the NTP bioassay, showing no carcinogenic activity in mice and no carcinogenic activity in rats except an increase of mononuclear cell leukaemia in female rats which is of doubtful relevance, the study presented here argues that paracetamol does not pose an unacceptable (if any) genotoxic/carcinogenic risk to man.

Phenobarbital: does the positive result in TA1535 indicate genotoxic properties?

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.

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice.

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.

Study of artificial flavouring substances for mutagenicity in the Salmonella/microsome, Basc and micronucleus tests.

Seventy-six compounds used as artificial flavouring substances in food products were studied for mutagenic properties by the use of the Salmonella/mammalian microsome test (Ames test), Basc test on Drosophila melanogaster and micronucleus test on mouse bone marrow. The following four compounds were mutagenic in Ames tests: ethyl nitrite, ethyl 3-phenylglycidate, 6-methylquinoline and musk ambrette. Of these, ethyl nitrite and musk ambrette also induced a significant (P less than or equal to 0.01) increase in sex-linked recessive lethal mutations in Drosophila. Two further compounds, ethyl 3-methyl-3-phenylglycidate and 4-n-propylanisole, appeared weakly mutagenic in Drosophila only. The result with 4-n-propylanisole was judged to be of equivocal biological significance. None of the flavouring substances induced micronuclei, i.e. cytogenetic damage in the bone marrow of mice.

5-Bromodeoxyuridine tablets with improved depot effect for analysis in vivo of sister-chromatid exchanges in bone-marrow and spermatogonial cells.

An improved 5-bromodeoxyuridine (BrdU) tablet technique for observation in vivo of SCE in mouse bone-marrow and spermatogonial cells is described. BrdU tablets were coated with agar as protecting barrier before subcutaneous implantation into mice. In comparison with the original tablets, the agar-coated tablets provided a slower and more uniform delivery of BrdU to the animals. This was corroborated (1) by recovering the undissolved portion of tablets at 1-2-h intervals, and (2) by quantitative determination of the BrdU levels in blood with the help of an analytical HPLC technique. The time required for complete dissolution of the coated tablets was considerably longer than that for the original tablets. This means that the dose of BrdU required for observation of SCE in mouse bone-marrow cells can be reduced accordingly. By using these modified tablets, therefore, undesired effects of high doses of BrdU on mutation (base-line SCE frequency) as well as on cellular replication and proliferation can be diminished. Moreover, the improved depot effect of the modified tablets facilitates the differential labeling of sister chromatids in mouse spermatogonia, a tissue containing cells with a relatively long DNA synthesis period.

Mutagenicity of cosmetics ingredients licensed by the European Communities.

As part of our investigation into mutagenic effects of environmental compounds, we studied chemicals allowed as ingredients of cosmetics according to the guidelines of the Council of the European Communities (27 July 1976). We used three systems, the Salmonella/microsome test, the Basc test on Drosophila and the micronucleus test on mouse bone marrow. Of the 31 chemicals tested, 15 were mutagenic in the Ames test; and of these, 5 were also mutagenic in the Basc test and 2 in the micronucleus test.

Mutagenicity study of Remsen-Fahlberg saccharin and contaminants.

Saccharin and contaminants of commercial Remsen-Fahlberg saccharin were studied for mutagenic potential with the use of the Salmonella/microsome test, Basc-test in Drosophila melanogaster and micronucleus test in mice. In none of these tests were mutagenic effects of saccharin observed. Likewise, the ortho- and para-sulfamoylbenzoic acids (OSBA and PSBA) were ineffective. Para-toluenesulfonamide (PTS) and the major contaminant ortho-toluene-sulfonamide (OTS) exhibited weak mutagenic effects in a modified Salmonella/microsome test and in Drosophila. These results do not indicate mutagenic and therewith correlated carcinogenic potential of saccharin, but they emphasize the possible activity of contaminants.

Synthesis of inducible enzymes in irradiated yeast cells: inhibition by ionizing radiation.

The induced activity of the enzyme arginase was measured in diploid wild type Saccharomyces cerevisiae after X-ray and 241Am-alpha-particle exposure. It was found that after doses which are comparable to those necessary to reduce survival, little effect on enzyme activity is seen immediately after irradiation but it is reduced on further incubation. In this case there is an oxygen enhancement ratio of about 2 as for survival. Suppression immediately after exposure requires considerably higher doses, and no oxygen effect is seen. To achieve the same effect with alpha-particles requires even higher doses, the apparent r.b.e. is about 0 . 1. X-ray damage to induced enzyme activity is subject to liquid holding recovery. The results are discussed in relation to current theories of gene inactivation by ionizing radiation.