Elmar Gocke

Recommendations for the performance of bacterial mutation assays

At the International Workshop on the Standardisation of Genotoxicity Test Procedures, in Melbourne (27-28 February 1993), the current international guidelines for the correct conduct of bacterial mutation assays were considered, and the major differences between them were examined. An attempt was made to construct a scientifically based, internationally harmonized protocol. The main points of agreement were as follows. The consensus opinion was that there are currently insufficient data to justify a preference for either the preincubation or plate-incorporation methodologies as the initial test. Whichever method is used there was consensus agreement that the bacterial test battery should consist of S. typhimurium TA1537, TA1535, TA98 and TA100. There was also consensus that the 3 strains TA97a, TA97 and TA1537 could be used interchangeably. Although it was not possible to achieve a consensus, the majority of the working group members agreed that strains for the detection of mutagens acting specifically on AT base pairs should be routinely included within the test battery. These strains may be S. typhimurium TA102 or E. coli WP2 strains (WP2 pKM101 and WP2 uvrA or WP2 uvrA pkM101). With regard to study design it was universally agreed that 5 doses of test compound should be used in each experiment, and a majority agreement was obtained for 3 plates per dose. The use of 2 plates per dose is acceptable ONLY if the experiment is repeated. It is recommended that the negative controls may consist of solvent control alone provided that historical data are available to demonstrate lack of effect of the solvent in question. Positive control compounds should be included in all experiments, although the nature of these control compounds need not be specified in the guidelines. There was consensus agreement that for non-toxic freely soluble test agents, an upper limit of 5 mg/plate should be tested (5 microliters per plate for liquids). For insoluble or toxic compounds, the recommendations were the same as those for other in vitro tests (see appropriate paper). A consensus agreement was reached on the need to carry out further tests if equivocal results are obtained in the initial test, although it was generally agreed that the design of the repeat study should be left flexible. As there are little or no data to support the use of an exact repeat assay, a majority of the group recommended that negative results in the first test should be further investigated by either conducting a modified repeat (e.g. S9 titration) or by conducting the alternative methodology.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Oxidative DNA damage and mutations induced by a polar photosensitizer, Ro19-8022

The oxidative DNA damage induced by the polar photosensitizer Ro19-8022 in the presence of light was studied and correlated with the associated mutagenicity. Both in isolated DNA and AS52 Chinese hamster ovary cells, photoexcited Ro19-8022 gave rise to a DNA damage profile that was similar to that caused by singlet oxygen: base modifications sensitive to the repair endonuclease Fpg protein, which according to high-performance liquid chromatography (HPLC) analysis were predominantly 8-hydroxyguanine (8-oxoG) residues, were generated in much higher yield than single-strand breaks, sites of base loss (AP sites) and oxidative pyrimidine modifications sensitive to endonuclease III. Fifty percent of the Fpg-sensitive modifications were repaired within 2 h. Under conditions that induced 10 Fpg-sensitive modifications per 10(6) bp (six 8-oxoG residues per 10(6) bp), approximately 60 mutations per 10(6) cells were induced in the gpt locus of the AS52 cells. A rather similar mutation frequency was observed when a plasmid carrying the gpt gene was exposed to Ro19-8022 plus light under cell-free conditions and subsequently replicated in bacteria. Sequence analysis revealed that GC-->TA and GC-->CG transversions accounted for 90% of the base substitutions. A significant generation of micronuclei was detectable in AS52 cells exposed to the photosensitizer plus light as well.

Mutagenicity studies with x-ray-contrast media, analgesics, antipyretics, antirheumatics and some other pharmaceutical drugs in bacterial, Drosophila and mammalian test systems.

As part of our investigation into mutagenic effects of environmental compounds, we studied 21 pharmaceuticals most frequently sold in West Germany: 6 X-ray-contrast media, 13 analgesics, antipyretics and antirheumatics, 1 central stimulant, and 1 antidepressant. They were studied in different bacterial, Drosophila and mammalian test systems. 4 of these 21 compounds could be detected as mutagens in one of the test systems. namely: 1,2-dichloroethane induced an increase in the frequency of recessive sex-linked lethal mutations in Drosophila melanogaster, quinine dihydrochloride and dimethylaminophenazone were mutagenic in the Salmonella typhimurium tester strain TA98 in the presence of S-9 liver fraction derived from Aroclor-induced rats, and trilithium citrate caused a significant effect in the micronucleus test on bone marrow of NMRI mice.

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.

Mechanism of quinolone mutagenicity in bacteria

Gyrase inhibitors of the quinolone type are genotoxic in bacteria. A functioning excision-repair system is needed to show the mutagenic activity in the Ames tester strains. The antibiotics do not interact with DNA directly but induce the gyrase enzyme to cleave the DNA with protein covalently bound at the site-specific double-strand scission. A prominent site of action is shown to be located at a distance of about 80 bp from the hisG428 sequence at which mutations are scored in the responsive strain TA102. Since the interaction of quinolones with the mammalian counterparts of gyrase, topoisomerase II, is lower by orders of magnitude, it is argued that the bacterial screening tests are of little relevance for predicting effects in mammalian cells.

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.

Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate.

In order to assess the risk of patients being exposed to an anti-AIDS medication contaminated with EMS we have performed in depth genotoxicity, general toxicity and DMPK investigations. The results of these studies are reported in the accompanying papers of this issue. Prior to starting our investigations we searched the literature for toxicity data on this well established mutagen with specific attention to dose-response relations in in vivo genotoxicity studies, since, obviously, in vivo data are pivotal for risk assessment. There are numerous published in vivo genotoxicity studies on EMS, with generally 50mg/kg - or higher - being the minimal dose used. The dose of 50mg/kg induced effects in some, but not all studies, while the dose of 100mg/kg was clearly positive in most studies, except for heritable mutations where a single dose of 100mg/kg was not observed to induce measurable effects in post-meiotic stages and even the maximal dose of 250 mg/kg was negative in pre-meiotic stages of male germ cell development. For somatic cells, NOEL values could not be derived for any of the endpoints studied. Although a large number of genotoxicity studies are available, none of the studies was sufficiently detailed to allow unambiguous conclusions about the presence of a (practical) threshold. But in most cases the dose-responses show a sublinear relationship (i.e. the slope increases with dose) which indicates that the data would not be incompatible with a threshold dose-response relationship. This stands in contrast to data on ethylnitrosourea (ENU) which has been studied concommittantly with EMS in several in vitro and in vivo genotoxicity investigations. ENU generally appeared to induce genotoxic effects with linear dose relationships. We also review the more limited data reported on teratogenicity and carcinogenicity of EMS. Induction of fetal malformations in mice appeared to have a NOEL of 100mg/kg. Classical life-time carcinogenicity studies have not been performed with EMS. Induction of mammary, lung, kidney, brain, and liver tumors has been observed after various short term treatment regimes. In none of the published studies a no effect level was reported and no exposure data are available. Overall, the experimental data do not fully characterize the carcinogenic potential of EMS and are insufficient for a risk extrapolation to humans. Although the data on teratogenicity and carcinogenicity are insufficient for assessing dose-response relations it is generally accepted that the genotoxic property of EMS is at the base of the teratogenic and carcinogenic effects.

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.

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