Automated microbial mutagenicity testing with COBAS BACT®

Mutation Research, 164 (1986) 287-299 Elsevier

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Gesellschaft fi r Umwelt-Mutationsforschung e.V. (GUM) A German-speaking Section of the European Environmental Mutagen Society (EEMS) Abstracts of papers presented at the 9th meeting, 16-18 October 1985, Miinchen-Neuherberg (F.R. G.) (Received 5 March 1986) (Accepted 12 March 1986)

1 Abel, G., and W. G~3ggelmann, Institut fi~r Botanik und Pharmazeutische Biologie, Universitat Erlangen-Ni~rnberg, D-8520 Erlangen, and Institut fflr Toxikologie, GSF Miinchen, D-8042 Neuherberg (F.R.G.)

Genotoxic activity of 13-asarone and commercial calamus drugs fl-Asarone is a characteristic component of the volatile oil of calamus. In 1967, a toxicity study in rats showed the carcinogenic effect of Indian calamus oil which contains about 80% fl-asarone. This carcinogenic effect could be ascribed to flasarone. We investigated fl-asarone in human lymphocytes in vitro for the induction of structural chromosome aberrations, fl-Asarone had a clastogenic effect after metabolic activation by rat liver microsomes. The microsomal protein content influences the chromosome breakage rate showing multiply damaged metaphases with several translocations. In addition, fl-asarone inhibited the growth of lymphocytes. Measurements of D N A content with the flow-cytometer were used to determine in which cell cycle phases the lymphocytes were inhibited or blocked by fl-asarone. In human therapy preparations of calamus roots are used. In addition to calamus, commercial drugs contain extracts of other medicinal plants and are recommended for the treatment of stomach disorders. 6 commercial calamus drugs were examined in the Ames test. In the presence of $9

mix the numbers of induced revertants in strain TA100 increased with doses of 50-300 /~1 drug/plate. Corresponding experiments in human lymphocytes are under investigation. With regard to increased drug safety the amount of B-asarone in pharmaceutically applied calamus drugs should be limited. 2 Arni, P., and D. Mi~ller, Ciba-Geigy Ltd., CH-4002 Basle (Switzerland)

Automated microbial mutagenicity testing with COBAS BACY ® If carried out in accordance with the current guidelines of EC and OECD, the Salmonella/ mammalian-microsome mutagenicity test developed by Prof. Ames requires great expenditure in terms of material and labour. Thus, it appeared desirable to modify the test system, permitting automated mutagenicity testing. This can be accomplished by using the COBAS BACT ®, an automatic microbial analyser, developed by Roche. The test system is based on the principle of turbidity measurement. 106-107 bacteria/ml of histidine-requiring strains of Salmonella typhimurium are incubated in Vogel-Bonner medium E, containing 0.4% nutrient broth (Difco) or 0.4 /~g/ml L-histidine, together with the test material and, if required, with $9 mix in measuring chambers with a volume of 300 /~1. At intervals of 20 rain turbidity measurement took place over a period of approx. 46 h. The growth of back-

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mutants results in an enhancement of turbidity of the medium, which is detectable after 18 h or later. The number of introduced bacteria and the quality of the traces of nutrient medium added to the Vogel-Bonner medium E strongly influence the background growth of the histidine-auxotrophic bacteria and subsequently also the generation of spontaneous and induced mutants. Depending on the concentration or on the potency of a mutagen, the rise of the growth curve of mutants occurs at an earlier or a later point of time. With strain TA1537 and 9-aminoacridine in a concentration of 2.6 /~g/ml, revertant growth was visible 29 h after the start of the experiment. Less than 10 m u t a n t s / c h a m b e r were estimated to be induced in this case. At the concentration of 8.0 /2g/ml, mutant growth was visible 23 h after the beginning of the experiment. This corresponds to approximately 1000 induced m u t a n t s / m e a s u r i n g chamber. In a validation study with over 30 chemicals a good correlation between results of the A m e s test and the COBAS BACT ~ system was demonstrated. The COBAS BACT ® system proved to be at least as sensitive as the Ames test. Thioacetamide and safrole, both carcinogenic in animal studies but negative in the standard Ames test, showed positive results in the COBAS BACT ® system. The test system thus may be used as a substitute for the Ames test. It offers additional valuable information on the kinetics of growth of auxotrophic and prototrophic bacteria during the experiment. 3 Basler, A., Bundesgesundheitsamt, Postfach 33 00 13, D-1000 Berlin 33 (F.R.G.)

Mutagenicity and carcinogenicity of formaldehyde Formaldehyde exposure may affect health. In man, irritations of mucosae and allergies have been documented. Furthermore, formaldehyde is suspected of having a carcinogenic potential. In chronic animal experiments, species-specific reactions of the mucosae of the respiratory tract were seen. Lifelong exposure to high formaldehyde concentrations produced chronic ulceration of the nasal mucosa, severe dyspnea and premature death

of animals. This concentration (14.3 ppm) resulted also in the development of squamous cell carcinoma in the nasal cavities of rats (Swenberg et al., Cancer Res.+ 40 (1980) 3398 3402). It is a matter of discussion whether the maintenance of severe ulceration is an essential step in the multi-stage process of the development of formaldehyde-induced nasal cell carcinoma. On the other hand, it might be possible that mutations induced at non-toxic concentrations are the initiating step. According to the finding that formaldehyde induces mutations in bacteria, fungi and Drosophila, as well as in a variety of mammalian cells in vitro (reviewed by Auerbach et al., Mutation Res., 39 (1977) 317-362), it has been assumed that formaldehyde interacts with the genetic material in the mucosa of exposed mammals. However, mutagenicity tests performed with mammals in vivo following exposure by inhalation yielded negative results. To clarify the influence of metabolizing enzymes on formaldehyde genotoxicity in vitro, experiments were performed in the absence and presence of different exogenous metabolizing systems, i.e. $9 mix and primary rat hepatocytes (Basler et al., Arch. Toxicol., 58 (1985) 10-13). In these experiments, formaldehyde induced SCE in V79 cells in the absence of a metabolizing system in a dose- and exposuretime-dependent manner. In contrast, the number of formaldehyde-induced SCE decreased in the presence of an exogenous metabolizing system. $9 mix as well as hepatocytes reduced the SCE frequency nearly to that of the control range. It could be demonstrated that the reduction was not due to an unspecific binding of formaldehyde to macromolecules of the added $9 mix. The decrease in genotoxic effects, due to rapid metabolization of formaldehyde explains the differences between results obtained in the in vitro experiments - - performed without metabolizing systems - - and in vivo results. The main metabolite of formaldehyde, formic acid, did not show any SCE-inducing effects. Formaldehyde is rapidly converted to formiate in vivo in many tissues. Since there are formaldehyde-metabolizing enzymes in the nasal mucosa of the rat too, it is unlikely that mutations are induced in this tissue at low concentrations without overloading the protective mechanisms such as